Amino acid substituted molecules

ABSTRACT

The invention provides compositions and methods of identifying, modifying and producing modified target molecules, including therapeutic molecules by modification with non-natural amino acids. Certain aspects of the invention include methods of adding a chemical moiety to a target molecule, and the compositions resulting therefrom. Certain aspects of the invention also relate to kits for identifying, modifying and producing modified target molecules described herein.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/509,213, filed Jul. 24, 2009, which is a continuation application ofU.S. patent application Ser. No. 12/271,747, filed Nov. 14, 2008, whichis a continuation-in-part application of U.S. patent application Ser.No. 12/105,154, filed Apr. 17, 2008, which is a continuation-in-partapplication of U.S. patent application Ser. No. 11/743,608, filed on May2, 2007; and a continuation-in-part application of U.S. patentapplication Ser. No. 12/105,155, filed Apr. 17, 2008, which is acontinuation-in-part application of U.S. patent application Ser. No.11/743,608, filed on May 2, 2007; and claims the benefit of the filingdate of U.S. Provisional Application 61/190,035, filed Aug. 20, 2007,U.S. Provisional Application 60/796,752, filed on May 2, 2006, U.S.Provisional Application 60/796,907, filed on May 2, 2006, and U.S.Provisional Application 60/796,701, filed on May 2, 2006; all of theseapplications are incorporated herein by reference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 110197_(—)410C6 SEQUENCE LISTING.txt. The textfile is 16 KB, was created on Nov. 2, 2009, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Molecules, including proteins, may be engineered through modification ofthe structural, catalytic and/or binding properties, as well as for thede novo design of artificial molecules. Molecular or protein engineeringrelies on an efficient recognition mechanism for incorporating desiredamino acid residues in specifically chosen locations of the proteinsequence or structural region. This process has been very useful fordesigning new macromolecules with precise control of composition andarchitecture; however, a major limitation exists when the mutagenesis isrestricted to the 20 naturally occurring amino acids. For this reason,it is becoming increasingly clear that incorporation of non-naturalamino acids can extend the scope and impact of molecular and proteinengineering methods. Thus, for many applications of designedmacromolecules, it would be desirable to develop methods forincorporating amino acids that have novel chemical functionality notpossessed by the 20 amino acids commonly found in naturally occuringproteins, or to utilize a non-natural amino acid residue for ananchoring position for further chemical or biological modification.

For example, if certain changes in a protein or other molecule aredesired (such as the size, acidity, nucleophilicity, hydrogen-bonding orhydrophobic properties, or other properties of amino acids) to fulfill aspecific structural or functional property of interest, it would beadvantageous to incorporate non-natural amino acid residues into themolecule. Such an advantage would greatly expand the ability torationally and systematically manipulate the structures of proteins, inorder to probe protein function, modify existing proteins, and createartificial proteins with new properties.

2. Description of the Related Art

Proteins are synthesized through a process beginning with RNAtranscription from DNA, followed by protein translation in the cell. Inorder for translation to occur, a ribosome binds to a messenger RNA(mRNA) that has been transcribed from DNA. During translation, eachtransfer RNA (tRNA) is matched with its cognate amino acid by acollection of enzymes called aminoacyl-tRNA synthetases (AARS). The AARScharge each tRNA with the appropriate amino acid, thereby facilitatingtranslation of the mRNA. As the process continues, the protein iselongated by the addition of the amino acids by the AARS.

Most cells make twenty different AARS, each corresponding to one of thetwenty naturally occurring amino acids. The AARS enzymes functionoptimally with its own cognate amino acid and set of tRNA moleculesappropriate to that amino acid.

Proteins may be modified or synthesized de novo through proteinengineering techniques. In particular, proteins may be altered ormodified to delete, substitute or add amino acids or modify existingamino acids. For example, it may be desirable to change at least oneparticular characteristic of a protein in order to develop a novelchemical functionality. Such characteristics may include the size,acidity, nucleophilicity, hydrogen-bonding or hydrophilic properties ofcertain amino acids in a protein.

Modifying molecules, including proteins, is presently largelyinefficient and ineffective, with large batch-to-batch variations inquality and quantity produced. In this regard, it would be beneficial todevelop an efficient method for designing molecules, including proteins,with improved properties and attached chemical moieties. The presentinvention provides such an advantage, as well as many others that areexpressed or implied in the present disclosure.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods, compositions (includingpharmaceutical compositions) as well as kits of various embodimentsdisclosed herein. More specifically, the present invention relates tomethods, compositions and kits relating to modified molecules comprisingone or more amino acid substitutions or additions with a naturallyoccurring amino acid (generally, an amino acid that is different thanthe one occurring in the native polypeptide sequence), one or more aminoacid substitutions with a non-naturally occurring amino acid, and achemical moiety added to said non-natural amino acid residue.

Some aspects of the disclosure relate to a method for modifying amolecule comprising one or more rounds of the steps of: (a) substitutingone or more amino acid residues in said molecule with a differentnaturally occurring amino acid residue; and (b) substituting one or moreamino acid residues with a non-natural amino acid residue wherein saidmolecule retains a native function. Amino acid residue position orlocation that may be substituted with a non-natural amino acid includethe amino terminus of the molecule. Other positions that may be havenon-natural amino acids incorporated include surface exposed or solventexposed locations in the target molecule's native structure which do notresult in loss of function. In certain aspects, adding one or morenaturally occurring amino acid residues to said molecule is conductedprior to substituting said one or more naturally occurring amino acidresidues with a non-natural amino acid residue. In certain aspects, theone or more amino acid residues substituted in step (a) is located inthe same amino acid position in the molecule as the one or more aminoacid residues substituted in step (b). In other aspects, the one or moreamino acid residues substituted in step (a) is located in a differentamino acid position in the molecule as the one or more amino acidresidues substituted in step (b).

In certain embodiments, a chemical moiety is added to said one or morenon-natural amino acid residues. In other embodiments, the nativefunction of the molecule is equal to or greater in magnitude compared tothe function of a corresponding wild type molecule.

In certain embodiments, one or more amino acid residues substituted instep (a) comprises approximately less than or equal to fifteen, lessthan or equal to ten, less than or equal to eight, less than or equal tosix, less than or equal to four, less than or equal to three, less thanor equal to two, less than or equal to one amino acid residue(s). Incertain embodiments, the one or more amino acid residues substituted instep (b) comprises approximately less than or equal to fifteen, lessthan or equal to ten, less than or equal to eight, less than or equal tosix, less than or equal to four, less than or equal to three, less thanor equal to two, less than or equal to one amino acid residue(s). Incertain aspects, the one or more residues substituted in step (a) or (b)comprise amino acid residues from a single amino acid family ordifferent amino acid families. In some embodiments, the one or moreamino acid residues substituted in step (a) or (b) compriseapproximately one, two, three, four, five, six, seven, eight, nine, ten,or more amino acid residues from the same amino acid family.

In certain aspects, said one or more amino acid residues is selectedfrom the group consisting of: alanine, arginine, aspartic acid,glutamine, glutamic acid, glycine, praline, serine, leucine, cysteine,valine, lysine, methionine, tryptophan, phenylalanine, arginine,tyrosine, threonine, isoleucine, histidine, lysine and asparagine. Someaspects further comprise adding a chemical moiety to said non-naturalamino acid residue. In some aspects, the chemical moiety is selectedfrom the group consisting of: cytotoxins, pharmaceutical drugs, dyes orfluorescent labels, a nucleophilic or electrophilic group, a ketone oraldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide,a polypeptide, a protein, an oligosaccharide, poly(ethylene) glycol withany molecular weight and in any geometry, polyvinyl alcohol, metals,metal complexes, polyamines, imidizoles, carbohydrates, lipids,biopolymers, particles, solid supports, a polymer, a targeting agent, anaffinity group, any agent to which a complementary reactive chemicalgroup can be attached, biophysical or biochemical probes,isotypically-labeled probes, spin-label amino acids, fluorophores, aryliodides and bromides. In some cases, the non-natural amino acid residueis fluorinated, electroactive or unsaturated.

In some embodiments, non-natural amino acid is selected from the groupconsisting of: azidohomoalanine, homoproparglyglycine,p-bromophenylalanine, p-iodophenylalanine, azidophenylalanine,acetylphenylalanine and ethynylephenylalanine.

In some embodiments the molecule is selected from the group consistingof: a peptide, polypeptide, protein, carbohydrate, deoxyribonucleicacid, ribonucleic acid, lipid, biopolymer or other molecule.

In other embodiments, the molecule may be a therapeutic, diagnostic, orother molecule selected from the group consisting of: an antibody,antibody fragment, antibody derivative, Fab, Fab′, F(ab)2, Fd, Fv, ScFv,diabody, tribody, tetrabody, dimer, trimer or minibody, a cytokine,Factor VII, Factor VIII, Factor IX, Follitropin, G-CSF, GM-CSF, GLP-1,human growth hormone, interferon-α, interferon-β, interferon-γ,interferon-Ω, interferon-τ, a transcriptional modulator that modulatescell growth, differentiation, or regulation, expression activator,inflammatory molecule, growth factor, growth factor receptor, andoncogene product.

In some aspects, one or more amino acid residues are substituted by atechnique selected from the group consisting of: chemical mutagenesis,site-directed mutagenesis, error-prone PCR, homologous recombination,gene shuffling, or by computational methods or by comparison of relatedgene sequences. Non-natural amino acids may be incorporated in theprotein using multi-site or site specific incorporation by a host cell.Further, the amino acid position at which the non-nautral amino acid isincorporated may be specified by a codon that is typically used tospecify a naturally occurring amino acid (such as a wobble codon, a biascodon, a sixth box codon, a 4 box codon, or any other sense codon thatthe host cell or in vitro translation system might be used to specifiy anon-natural amino acid incorporation site), or a codon which istypically a stop codon, such as amber, ochre, or opal, or a frameshiftcodon. In other aspects, the method may further comprise modifying apolynucleotide encoding said molecule.

In some embodiments, the method further comprises an in vivo or in vitrotranslational system. In some aspects, the translation system comprisesa host cell selected from the group consisting of: prokaryotic,eukaryotic, and insect cells.

Some aspects further comprise using structural coordinates of saidmolecule to derive one or more energy calculations in order to determinewhich one or more amino acid residues are energetically favorable tosubstitution with a different amino acid residue. Some energeycalculations that may be utilized include: forcefield calculation,original DEE or Goldstein DEE, Monte Carlo search, derived from arotamer library, derived from a ligand or receptor binding site of themolecule, derived from one or more salvation calculations, derived fromone or more binding energies, or HierDock computational screening.

In some embodiments the method further comprises using the identity ofthe penultimate amino acid residue in the molecule in order to determinewhich one or more amino acid residues may be efficiently substituted atthe amino terminus. In certain embodiments, the penultimate amino acidresidue is a non-natural amino acid and is either substituted or addedto the target molecule in order to either retain or remove thenon-natural amino acid residue at the first position of the aminoterminus of the polypeptide during processing (transcription,translation, and/or post-translational modifications).

Other aspects of the disclosure relate to a compositon comprising amodified molecule comprising one or more amino acid residues substitutedwith a different naturally occurring amino acid residue to make asequence that differs from the native sequence of the molecule; one ormore non-natural amino acid residues and a chemical moiety, wherein atleast one of the non-natural amino acid residues is located at the aminoterminus, and wherein said modified molecule retains a native function.Some embodiments include the composition wherein a native function isequal to or greater in magnitude compared to the function of acorresponding wild type molecule.

In some embodiments, the molecule comprises a chemical moiety selectedfrom the group consisting of: cytotoxins, pharmaceutical drugs, dyes orfluorescent labels, a nucleophilic or electrophilic group, a ketone oraldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide,a polypeptide, a protein, an oligosaccharide, polyethylene glycol withany molecular weight and in any geometry, polyvinyl alcohol, metals,metal complexes, polyamines, imidizoles, carbohydrates, lipids,biopolymers, particles, solid supports, a polymer, a targeting agent, anaffinity group, any agent to which a complementary reactive chemicalgroup can be attached, biophysical or biochemical probes,isotypically-labeled probes, spin-label amino acids, fluorophores, aryliodides and bromides.

The modified molecule may be a therapeutic, diagnostic, or othermolecule selected from the group consisting of: an antibody, antibodyfragment, antibody derivative, Fab, Fab′, F(ab)2, Fd, Fv, ScFv, diabody,tribody, tetrabody, dimer, trimer or minibody, a cytokine, Factor VII,Factor VIII, Follitropin, G-CSF, GM-CSF, growth hormone, erythropoietin,thrombopoietin, interferon-α, interferon-β, interferon-γ, interferon-Ω,interferon-τ, GLP-1, a transcriptional modulator that modulates cellgrowth, differentiation, or regulation, expression activator,inflammatory molecule, growth factor, growth factor receptor, andoncogene product.

In some embodiments, the molecule comprises interferon-β. In someembodiments, the naturally occurring residues 1, 2, 36, 40, 44, 62, or117, of the interferon-β or any combination thereof, is altered toanother amino acid residue. In certain embodiments, any one or more ofthose residues may be replaced with azidohomoalanine,para-bromophenylalanine, homoproparglyglycine, ethynylphenylalanine,azidophenylalanine, or para-iodophenylalanine. In certain embodiments,the non-natural amino acid residue is located at a terminal end of themolecule. In some cases, the terminal end comprises the amino terminus.In some cases, the terminal end comprises the carboxyl terminus.

In certain embodiments, the one or more amino acid residues substitutedwith another naturally occurring amino acid residue comprisessubstituting methionine at residue 62 of human interferon β toisoleucine, and/or isoleucine at residue 40 of human interferon β tophenylalanine, and/or isoleucine at residue position 44 of humaninterferon β to leucine. In some embodiments, the methionine at position117 of human interferon β is substituted. In some cases, the methionineat position 117 is substituted with serine or threonine. In someembodiments, the methionine at position 36 is substituted withthreonine, isoleucine, or alanine. In any of these embodiments, thenaturally occurring amino acid residues at the aforementioned positionsmay be substituted with non-natural amino acids, includingazidohomoalanine, homoproparglyglycine, p-bromophenylalanine,azidophenylalanine, acetylphenylalanine, ethynylphenylalanine,azidophenylalanine, or p-iodophenylalanine. In addition, any of thenon-natural amino acids may further comprise a chemical moiety(including polyethylene glycol).

One specific embodiment is a modified interferon-β having the followingsubstitutions of naturally occurring residues within human interferon β:methionine 36 is substituted by isoleucine; methionine 62 is substitutedby isoleucine; methionine 117 is substituted by threonine; isoleucine 40is substituted by phenylalanine; isoleucine 44 is substituted byleucine; and serine 2 is substituted by glutamic acid. Anotherembodiment comprises these substitutions, as well as substitution ofmethionine 1 with azidohomoalanine (AHA). In particular embodiments, thepresent invention includes a modified human interferon-β having asequence provided in SEQ ID NO:20 or 21, or a functional fragmentthereof.

In another embodiment, the modified molecule comprises human growthhormone and one or more amino acid residues to be substituted comprisetryptophan, phenylalanine, or methionine. In another embodiment, themolecule comprises G-CSF, erthyropoietin, GLP-1, phenylalaninehydroxylase, urikase, Factor VII, or follitropin.

Still other aspects relate to a pharmaceutical composition comprising amodified molecule comprising one or more amino acid residues substitutedwith a naturally occurring amino acid residue; and one or more residuessubstituted with one or more non-natural amino acid residue; and one ormore chemical moieties.

In certain embodiments, one or more properties of the molecule arealtered wherein said properties are selected from the group consistingof: toxicity, biodistribution, structural properties, spectroscopicproperties, chemical or photochemical properties, catalytic ability,serum half-life, shelf half-life, ability to react with toher moleculescovalently or non-covalently, stability, activity, conformation,substrate specificity, target binding affinity, antigen-binding ability,thermostability, resistance to at least one protease, tolerance to atleast one non-aqueous environment, glycosylation pattern,phosphorylation pattern, disulfide bonding, protease cleavage sitelocation, metal binding ability, co-factor binding ability,cross-linking ability, solubility, cysteinylation, deamidation,acetylation, biotinylation, oxidation, glutathionylation, sulphonation,immunogenicity, tissue penetration, fluorescence pegylation,multimerization ability, facility of purification, catalytic activity,vaccine stability, ability to function as a vaccine, redox potential,patient tolerance to a protein, increased efficacy of a protein in apatient, and improved delivery of a protein or protein product in apatient.

Thus, certain embodiments of the present invention relate to a methodfor producing a modified target polypeptide, comprising providing a hostcell, the host cell comprising a vector having a polynucleotide encodingthe target polypeptide, site-specifically incorporating one or morenon-natural amino acid codons into the polynucleotide, wherein at leastone non-natural amino acid codon corresponds to the first position ofthe amino terminus of the target polypeptide, (a) growing the host cellunder conditions such that the host cell expresses the targetpolypeptide, wherein the target molecule retains the non-natural aminoacid residue at the first position of the amino terminus, and whereinthe non-natural amino acid residue at the first position of the aminoterminus contains an azide, alkyne, vinyl, or aryl halide group, therebyproducing a modified target polypeptide.

In certain embodiments, one or more non-natural amino acid codon encodesthe penultimate position of the amino terminus of the targetpolypeptide. The methods may include one or more non-natural amino acidsis selected from the group consisting of: azidonorleucine,3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine,6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid,p-bromophenylalanine, p-idiophenylalanine, p-azidophenylalanine,3-(6-chloroindolyl)alanine, 3-(6-bromoindoyl)alanine,3-(5-bromoindolyl)alanine, azidohomoalanine, and p-chlorophenylalanine.

In certain embodiments, the target polypeptide is selected from thegroup consisting of: Factor VII, Factor VIII, Factor IX, Follitropin,thrombopoeitin, erythropoietin, human growth hormone, G-CSF, GM-CSF,interferon-α, interferon-β, interferon-γ, interferon-Ω, interferon-τ,and GLP-1.

In certain embodiments, the site-specifically incorporating one or moreamino acid codons is conducted by a technique selected from the groupconsisting of: site-directed mutagenesis, error-prone PCR, geneshuffling, homologous recombination, incorporation of an amber stopcodon, incorporation of a wobble codon, use of an external mutantaminoacyl-tRNA synthetase, and incorporation of a bias codon.

The present invention also relates to a composition comprising amodified target polynucleotide encoding a target polypeptide, the targetpolynucleotide comprising one or more non-natural amino acid codonswherein at least one non-natural amino acid codon contains an azide,alkyne, vinyl, or aryl halide group and corresponds to the firstposition of the amino terminus of the target polypeptide. In particularembodiments, the present invention includes a modified targetpolynucleotide that encodes a modified interferon-beta having a sequenceset forth in SEQ ID NO: 20 or 21. In related embodiments, the presentinvention includes a vector, e.g., an expression vector, comprising amodified target polynucleotide that encodes a modified interferon-betahaving a sequence set forth in SEQ ID NO: 20 or 21.

In certain embodiments, the composition further comprises a host cell.In particular embodiments, the host cell comprises a vector comprising amodified target polynucleotide of the present invention, e.g., amodified target polynucleotide that encodes a modified interferon-betahaving a sequence set forth in SEQ ID NO: 20 or 21.

In still other embodiments, the composition comprises at least onenon-natural amino acid codon corresponds to the penultimate position ofthe amino terminus of the target polypeptide. In still otherembodiments, the composition further comprises a chemical moietyattached to one or more non-natural amino acid residues in the targetpolypeptide. In still other embodiments, the composition comprises achemical moiety attached at least to the non-natural amino acid residuein the first position of the amino terminus of the target polypeptide.In some instances, the chemical moiety is covalently attached to thenon-natural amino acid corresponding to the first position of the aminoterminus of the target polypeptide. In other embodiments, the chemicalmoiety is attached to the non-natural amino acid corresponding to thefirst position of the amino terminus of the target polypeptide by asingle carbon-carbon linkage, a double carbon-carbon linkage, a triplecarbon-carbon linkage, or a triazole linkage between the chemical moietyand the non-natural amino acid. In still other embodiments, the chemicalmoiety is selected from the group consisting of: cytotoxins,pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic orelectrophilic group, a ketone or aldehyde, azide or alkyne compounds,photocaged groups, tags, a peptide, a polypeptide, a protein, anoligosaccharide, polyethylene glycol with any molecular weight and inany geometry, polyvinyl alcohol, metals, metal complexes, polyamines,imidizoles, carbohydrates, lipids, biopolymers, particles, solidsupports, a polymer, a targeting agent, an affinity group, any agent towhich a complementary reactive chemical group can be attached,biophysical or biochemical probes, isotypically-labeled probes,spin-label amino acids, fluorophores, aryl iodides and bromides.

The composition may include a modified target polypeptide is selectedfrom the group consisting of: an antibody, antibody fragment, antibodyderivative, Fab, Fab′, F(ab)2, Fd, Fv, ScFv, diabody, tribody,tetrabody, dimer, trimer or minibody, a cytokine, a transcriptionalmodulator that modulates cell growth, differentiation, or regulation,expression activator, inflammatory molecule, growth factor, growthfactor receptor, and oncogene product. The composition may be selectedfrom the group consisting of: Factor VII, Factor VIII, Factor IX,Follitropin, thrombopoeitin, erythropoietin, human growth hormone,G-CSF, GM-CSF, interferon-α, interferon-β, interferon-γ, interferon-Ω,interferon-τ, and GLP-1. Preferably, the composition comprisesinterferon-β. In certain embodiments, at least one of the non-naturalamino acid codons corresponds to positions selected from the groupconsisting of: 2, 17, 36, 40, 44, 62, and 117 of the modified targetpolypeptide.

Still other embodiments include a pharmaceutical composition comprisinga modified target polypeptide comprising a target polypeptide having oneor more non-natural amino acids residues incorporated, wherein at leastone of the non-natural amino acid residues corresponds to the firstposition of the amino terminus of the target polypeptide.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A illustrates in vitro biological activity of interferon-β mutantsin which the methionine at position 36 is substituted with anisoleucine, arginine, or threonine residue. Biological activity wasmeasured based on Daudi cell proliferation according to MTS metabolismafter 3 days exposure to interferon-β.

FIG. 1B illustrates in vitro biological activity of interferon-β mutantsin which the methionine at position 62 is substituted with a lysine,isoleucine, or valine residue. Biological activity was measured based onDaudi cell proliferation according to MTS metabolism after 3 daysexposure to interferon-β.

FIG. 1C illustrates the activity of interferon-β mutants in which themethionine at position 117 is substituted with threonine, tyrosine,serine, or glycine. HEK 293 cells were transfected with an interferon-βmutant, and supernatants collected at day 3. Interferon-β activity ofsupernatant or Avonex was measured based on inhibition of Daudi cellproliferation.

FIG. 1D illustrates the activity of interferon-β mutants in which themethionine at position 117 is substituted with a threonine, a mutant inwhich the methionine at position 62 is substituted with an isoleucine,the isoleucine at position 40 is substituted with phenylalanine, and theisoleucine at position 44 is substituted with leucine. AVONEX® (humaninterferon-β-1a) is manufactured by Biogen Idec, Inc. HEK 293 cells weretransfected with an interferon-β mutant, and supernatants collected.Interferon-β activity of the supernatant or Avonex was measured based onthe inhibition of Daudi cell proliferation.

FIG. 2 illustrates the activity of interferon-β mutants. Triple:methionine at position 62 is substituted with isoleucine, isoluecine atamino acid position 40 is substituted with phenylalanine, isoleucine atamino acid position 44 is substituted with leucine. WT: wild type, nomutations. Triple-M117S: methionine at position 62 is substituted withisoleucine, isoluecine at amino acid position 40 is substituted withphenylalanine, isoleucine at amino acid position 44 is substituted withleucine, and methionine at amino acid position 117 is substituted withserine. Triple-M117T: methionine at position 62 is substituted withisoleucine, isoluecine at amino acid position 40 is substituted withphenylalanine, isoleucine at amino acid position 44 is substituted withleucine, and methionine at position 117 is substituted with threonine.M36A-Triple: methionine at position 62 is substituted with isoleucine,isoluecine at amino acid position 40 is substituted with phenylalanine,isoleucine at amino acid position 44 is substituted with leucine, andmethionine at amino acid position 36 is substituted with alanine.M36T-Triple: methionine at position 62 is substituted with isoleucine,isoluecine at amino acid position 40 is substituted with phenylalanine,isoleucine at amino acid position 44 is substituted with leucine, andmethionine at position 36 is substituted with threonine. HEK 293 cellswere transfected with each mutant. Activity of interferon-β mutants wasmeasured based on inhibition of Daudi cell proliferation.

FIG. 3 illustrates the antiproliferative activity of variousinterferon-β mutants with a single methionine present in the molecule,and various mutations at the second amino acid position (S2). HEK 293cells were transfected with expression plasmids encoding the mutantconstructs (TT or TI), as indicated. TT indicates a mutant comprising atleast an M36T-M117T mutation or substitution. TI indicates a mutantcomprising at least an M36T-M117I mutation or substitution.

Sandwich ELISAs were performed on supernants and biological activity wasmeasured for rates of inhibition of proliferation.

FIG. 4 illustrates the antiviral activity of various interferon-βmutants with a single methionine present in the molecule, and variousmutations at the second amino acid position (S2). A549 cells wereincubated with EMC virus at a titer sufficient to give complete lysis ofthe cells in the absence of interferon. The supernatant was removed andcells were stained in crystal violet and buffered formalin. Cell countwas determined at OD₆₅₀ by a microplate reader.

FIG. 5 depicts two methionine analogs, azidohomoalanine andhomoproparglyglycine, as well as an activated poly(ethylene) glycolmolecule.

FIG. 6 illustrates an SDS-PAGE of pegylated interferon-β, with varioussized PEG molecules.

FIG. 7A illustrates antiviral activity for 10K-PEG interferon-βconjugate TIS2E. A549 cells were incubated with EMC virus at a titersufficient to give complete lysis of the cells in the absence ofinterferon. The supernatant was removed and cells were stained incrystal violet and buffered formalin. Cell count was determined at OD₆₅₀by a microplate reader.

FIG. 7B illustrates antiviral activity for various 20K-PEG interferon-βconjugates (TIS2E), according to standard procedures as described forFIG. 7A.

FIG. 8A illustrates ability of 10K-PEG interferon-β conjugate to inhibittumor progression as measured by tumor size of Daudi cells implantedinto SCID mice.

FIG. 8B illustrates ability of 20K-PEG interferon-β conjugate to inhibittumor progression as measured by tumor size of Daudi cells implantedinto SCID mice.

FIG. 9 illustrates in vivo potency of a representative PEG-IFNβ (atvarious levels to inhibit growth of Daudi cell tumors in scid mice atdifferent levels of administration, compared to buffer and Betaseron.

FIG. 10 illustrates antiviral activity for PEG-IFNβ. A549 cells wereincubated with EMC virus at a titer sufficient to give complete lysis ofthe cells in the absence of interferon. The supernatant was removed andcells were stained in crystal violet and buffered formalin. Cell countwas determined at OD₆₅₀ by a microplate reader.

FIG. 11 provides plasma levels of PEG-IFNβ in three cynomolgus monkeysfollowing subcutaneous administration of 0.3 mg/kg PEG-IFNβ.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention includes methods, compositions and kits foridentifying and/or modifying molecules, optionally testing the activityof the molecule, and/or purifying the molecule.

Specifically, some embodiments provide for modifying a molecule bydeletion of an amino acid and/or incorporation of one or morenon-natural amino acid residues into the molecule. In certainembodiments, at least the N-terminal amino acid (typically a methionine)is replaced with a non-natural amino acid. In certain other embodiments,a non-natural amino acid is incorporated at the penultimate position, inaddition to the N-terminal amino acid being replaced with a non-naturalamino acid, and possibly other non-natural amino acid incorporations inthe molecule. Certain embodiments utilize auxotrophic host cells forassistance in incorporating non-natural amino acids into the molecule.Certain other embodiments may utilize mutant transcription ortranslation machinery for assistance in incorporating non-natural aminoacids, while some embodiments will utilize both auxotrophic host cellsand mutant transcription or translation machinery. Exemplary means ofmutant transcription machinery include mutant tRNA and/or mutantamino-acyl tRNA synthetase(s). In some embodiments, a chemical moiety isattached to one or more of the non-natural amino acids of the modifiedmolecule.

Several detailed methods for altering molecules, including proteins, areset forth in U.S. patent application Ser. No. 09/620691, now abandoned;Ser. No. 10/851,564, pending as U.S. Publication No. 20040219488; Ser.No. 10/612,713, pending as U.S. Publication No. 20040058415; Ser. No.10/015,956, pending but not yet published; Ser. No. 11/094,625, pendingas U.S. Publication No. 20050260711; Ser. No. 11/130,583, pending asU.S. Publication No. 20050287639; U.S. Pat. No. 7,139,665; and U.S. Pat.No. 6,586,207; all of which are hereby incorporated by reference intheir entireties. Additionally, several issued U.S. patents discussmethods for calculating energy analysis for point mutations inmolecules, including proteins, such as U.S. Pat. Nos. 6,188,965;6,269,312; 6,708,120; 6,792,356; 6,801,861 and 6,804,611, all of whichare hereby incorporated by reference in their entireties. Any of thesereferenced, or any other methods of altering, modifying or identifyingmolecules may be used with the present invention.

Definitions

Before describing certain embodiments in detail, it is to be understoodthat this invention is not limited to particular compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular illustrative embodiments only, and is not intendedto be limiting. The terms used in this specification generally havetheir ordinary meanings in the art, within the context of this inventionand in the specific context where each term is used. Certain terms arediscussed below or elsewhere in the specification, to provide additionalguidance to the practitioner in describing the compositions and methodsof the invention and how to make and use them. The scope and meaning ofany use of a term will be apparent from the specific context in whichthe term is used. As such, the definitions set forth herein are intendedto provide illustrative guidance in ascertaining particular embodimentsof the invention, without limitation to particular compositions orbiological systems. As used in the present disclosure and claims, thesingular forms “a,” “an,” and “the” include plural forms unless thecontext clearly dictates otherwise.

“About” and “approximately,” as used herein, generally refer to anacceptable degree of error for the quantity measured, given the natureor precision of the measurements. Typical, exemplary degrees of errormay be within 20%, 10%, or 5% of a given value or range of values.Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, potentially within 5-fold or 2-fold of a given value.Numerical quantities given herein are approximate unless statedotherwise, meaning that the term “about” or “approximately” may beinferred when not expressly stated.

“Altered,” as used herein may be used synonymously with “changed,”“modified,” and in certain embodiments, “mutated” (e.g., a mutatedpolynucleotide may also be referred to as altered or modified).“Mutation” or “modification” generally refers to an alteration of atarget molecule, tRNA, or AARS that occurs at a nucleic acid level (i.e.altering a polynucleotide) rather than at an amino acid level (i.e.during fermentation). For example, a mutation or modification mayinclude any physical, chemical, or biological alteration or change tothe target molecule, typically at the genetic or nucleic acid level.

“Incorporation,” as used herein refers to any addition, substitution,replacement, mutation or other modification in which one or morenaturally occurring amino acid or non-natural amino acid is entered intothe target molecule in addition to or as a substitute for anothernaturally occurring amino acid or non-natural amino acid. As usedherein, “substitute” and any and all variations thereof, is synonomouswith “replace” and any and all variations thereof.

One of skill in the art would understand that a target molecule may bealtered by the addition, deletion, substitution, mutation, or chemicalmodification to any amino acid residue, amino acid group or component(e.g., amino acid side chain), or nucleic acid encoding an amino acidresidue in the target molecule. In certain embodiments described herein,a non-natural or other amino acid residue may be incorporated into atarget molecule by various methods, including but not limited tomodifying a codon of the polynucleotide to alter a naturally occurringamino acid to another naturally occurring amino acid, by altering thepolynucleotide from encoding a naturally occurring amino acid to anon-natural amino acid, or by adding a non-natural amino acid to themedia of the host cells during protein translation (fermentation)wherein the non-natural amino acid is utilized at a positioncorresponding to a codon specifiying a particular amino acid.

“Amino acid analog,” “non-canonical amino acid,” “unnatural amino acid,”“modified amino acid,” “unnatural AARS substrate,” “non-natural AARSsubstrate,” “non-standard amino acid,” “non-natural amino acid,”“unnatural amino acid,” and the like may all be used interchangeably,and is meant to include all amino acid-like compounds that are similarin structure and/or overall shape to one or more of the twenty L-aminoacids commonly found in naturally occurring proteins (Ala or A, Cys orC, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K,Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S,Thr or T, Val or V, Trp or W, Tyr or Y, as defined and listed in WIPOStandard ST.25 (1998), Appendix 2, Table 3). Amino acid analog can alsobe natural amino acids with modified side chains or backbones. Aminoacids can also be naturally occurring amino acids in D-, rather thanL-form. Preferably, these analogs usually are not “substrates” for theaminoacyl tRNA synthethases (AARSs) because of the normally highspecificity of the AARSs. Although occasionally, certain analogs withstructures or shapes sufficiently close to those of natural amino acidsmay be erroneously incorporated into proteins by AARSs, especiallymodified AARSs with relaxed substrate specificity. In a preferredembodiment, the analogs share backbone structures, and/or even the mostside chain structures of one or more natural amino acids, with the onlydifference(s) being containing one or more modified groups in themolecule. Such modification may include, without limitation,substitution of an atom (such as N) for a related atom (such as S),addition of a group (such as methyl, or hydroxyl group, etc.) or an atom(such as Cl or Br, etc.), deletion of a group (supra), substitution of acovalent bond (single bond for double bond, etc.), or combinationsthereof. Amino acid analogs may include α-hydroxy acids, and α-aminoacids, and can also be referred to as “modified amino acids,” or“unnatural AARS substrates.”

The amino acid analogs may either be naturally occurring or non-natural(e.g., synthesized). As will be appreciated by those in the art, anystructure for which a set of rotamers is known or can be generated canbe used as an amino acid analog. The side chains may be in either the(R) or the (S) configuration (or D- or L-configuration). In a preferredembodiment, the amino acids are in the (S) or L-configuration.

Preferably, the overall shape and size of the amino acid analogs aresuch that, upon being charged to (natural or modified or re-designed)tRNAs by (natural or re-designed) AARS, the analog-tRNA is a ribosomallyaccepted complex, i.e., the tRNA-analog complex can be accepted by theprokaryotic or eukaryotic ribosomes in an in vivo or in vitrotranslation system.

“Backbone,” or “template” includes the backbone atoms and any fixed sidechains (such as the anchor residue side chains) of the protein (e.g.,AARS).

“Protein backbone structure” or grammatical equivalents herein generallyrefers to the three dimensional coordinates that define the threedimensional structure of a particular protein. The structure thatcomprises a protein backbone structure (of a naturally occurringprotein) includes the nitrogen, the carbonyl carbon, the α-carbon, andthe carbonyl oxygen, along with the direction of the vector from theα-carbon to the β-carbon.

When the protein backbone structure is entered into a computer, it mayeither include the coordinates for both the backbone and the amino acidside chains, or just the backbone, i.e., with the coordinates for theamino acid side chains removed. If the former is done, the side chainatoms of each amino acid of the protein structure may be “stripped” orremoved from the structure of a protein, as is known in the art, leavingonly the coordinates for the “backbone” atoms (the nitrogen, carbonylcarbon and oxygen, and the α-carbon, and the hydrogens attached to thenitrogen and α-carbon).

Optionally, the protein backbone structure may be altered prior to theanalysis outlined below. In this embodiment, the representation of thestarting protein backbone structure is reduced to a description of thespatial arrangement of its secondary structural elements. The relativepositions of the secondary structural elements are defined by a set ofparameters called supersecondary structure parameters. These parametersare assigned values that can be systematically or randomly varied toalter the arrangement of the secondary structure elements to introduceexplicit backbone flexibility. The atomic coordinates of the backboneare then changed to reflect the altered supersecondary structuralparameters, and these new coordinates are input into the system for usein the subsequent protein design automation. See, for example, U.S. Pat.No. 6,269,312, hereby incorporated by reference in its entirety.

“Conformational energy” refers generally to the energy associated with aparticular “conformation,” or three-dimensional structure of amacromolecule, such as the energy associated with the conformation of aparticular protein. Interactions that tend to stabilize a protein haveenergies that are represented as negative energy values, whereasinteractions that destabilize a protein have positive energy values.Thus, the conformational energy for any stable protein is quantitativelyrepresented by a negative conformational energy value. Generally, theconformational energy for a particular protein will be related to thatprotein's stability. In particular, molecules that have a lower (i.e.,more negative) conformational energy are typically more stable, e.g., athigher temperatures (i.e., they have greater “thermal stability”).Accordingly, the conformational energy of a protein may also be referredto as the “stabilization energy.”

Typically, the conformational energy is calculated using an energy“force-field” that is able to calculate or estimate the energycontribution from various interactions dependent upon the conformationof a molecule. The force-field is comprised of terms that include theconformational energy of the α-carbon backbone, side chain—backboneinteractions, and side chain-side chain interactions. Typically,interactions with the backbone or side chain include terms for bondrotation, bond torsion, and bond length. The backbone-side chain andside chain-side chain interactions include van der Waals interactions,hydrogen-bonding, electrostatics and solvation terms. Electrostaticinteractions may include Coulomb interactions, dipole interactions andquadrapole interactions, as well as other similar terms.

Force-fields that may be used to determine the conformational energy fora polymer are well known in the art and include the CHARMM (see, Brookset al, J. Comp. Chem. 1983,4:187-217; MacKerell et al., in TheEncyclopedia of Computational Chemistry, Vol. 1:271-277, John Wiley &Sons, Chichester, 1998), AMBER (see, Cornell et al., J. Amer. Chem. Soc.1995, 117:5179; Woods et al., J. Phys. Chem. 1995, 99:3832-3846; Weineret al., J. Comp. Chem. 1986, 7:230; and Weiner et al., J. Amer. Chem.Soc. 1984, 106:765) and DREIDING (Mayo et al., J. Phys. Chem. 1990,94-:8897) force-fields, as well as others, all of which are herebyincorporated by reference.

In at least one embodiment, the hydrogen bonding and electrostaticsterms are as described in Dahiyat & Mayo, (Science 1997 278:82), herebyincorporated by reference in its entirety. The force field can also bedescribed to include atomic conformational terms (bond angles, bondlengths, torsions), as in other references. See, e.g., Nielsen J E,Andersen K V, Honig B, Hooft R W W, Klebe G, Vriend G, & Wade R C,Protein Engineering, 12: 657-662 (1999); Stikoff D, Lockhart D J, SharpK A & Honig B, Biophys. J., 67: 2251-2260 (1994); Hendscb Z S, Tidor B,Protein Science, 3: 211-226 (1994); Schneider J P, Lear J D, DeGrado WF, J. Am. Chem. Soc., 119: 5742-5743 (1997); Sidelar C V, Hendsch Z S,Tidor B, Protein Science, 7: 1898-1914 (1998), Jackson S E, Moracci M,Mastry N, Johnson C M, Fersht A R, Biochem., 32: 11259-11269 (1993);Eisenberg, D & McLachlan A D, Nature, 319: 199-203 (1986); Street A G &Mayo S L, Folding & Design, 3: 253-258 (1998); Eisenberg D & Wesson L,Protein Science, 1: 227-235 (1992); all of which are hereby incorporatedby reference in their entireties.

“Coupled residues” generally refers to residues in a molecule thatinteract through any mechanism. The interaction between the two residuesis therefore referred to as a “coupling interaction.” Coupled residuesgenerally contribute to polymer fitness through the couplinginteraction. Typically, the coupling interaction is a physical orchemical interaction, such as electrostatic interaction, van der Waalsinteraction, hydrogen bonding interaction, or a combination thereof. Asa result of the coupling interaction, changing the identity of eitherresidue will affect the “fitness” of the molecule, particularly if thechange disrupts the coupling interaction between the two residues.Coupling interaction may also be described by a distance parameterbetween residues in a molecule. If the residues are within a certaincutoff distance, they are considered interacting.

“Fitness” is used herein to generally denote the level or degree towhich a particular property or combination of properties for a molecule(such as a protein) are optimized. In certain embodiments of theinvention, the fitness of a protein may be determined by particularproperties that a user desires to improve. Thus, for example, thefitness of a protein may refer to the protein's thermal stability,structural stability, pharmaceutical capability, catalytic activity,ability to function as a vaccine, binding affinity, solubility (e.g., inaqueous or organic solvent), substrate specificity, resistance to atleast one protease, tolerance to at least one non-aqueous environmentand other activities. Other examples of fitness properties includeenantioselectivity, activity towards non-natural substrates, andalternative catalytic mechanisms. Coupling interactions can be modeledas a way of evaluating or predicting fitness. Fitness can be determinedor evaluated experimentally or theoretically, e.g., computationally.

Preferably, the fitness is quantitated so that each molecule, e.g., eachamino acid, will have a particular “fitness value”. For example, thefitness of a protein may be the rate at which the protein catalyzes aparticular chemical reaction, or the fitness may be the protein'sbinding affinity for a ligand. In a particularly preferred embodiment,the fitness of a protein refers to the conformational energy of thepolymer and is calculated, using any method known in the art. (See,e.g., Brooks B. R., Bruccoleri R E, Olafson, B D, States D J,Swaminathan S & Karplus M, J. Comp. Chem., 4: 187-217 (1983); Mayo S L,Olafson B D & Goddard W A G, J. Phys. Chem., 94: 8897-8909 (1990); PaboC O & Suchanek E G, Biochemistry, 25: 5987-5991 (1986), Lazar G A,Desjarlais J R & Handel T M, Protein Science, 6: 1167-1178 (1997); Lee C& Levitt M, Nature, 352: 448-451 (1991); Colombo G & Merz K M, J. Am.Chem. Soc., 121: 6895-6903 (1999); Weiner S J, Kollman P A, Case D A,Singh U C, Ghio C, Alagona G, Profeta S J, Weiner P, J. Am. Chem. Soc.,106: 765-784 (1984), Datta, et al., Protein Science 13: 2693-2705(2004), all of which are hereby incorporated by reference in theirentireties).

In at least one embodiment, the fitness of a protein is quantitated sothat the fitness value increases as the property or combination ofproperties is optimized. For example, in an embodiment where the thermalstability of a protein is to be optimized (conformational energy ispreferably decreased), the fitness value may be the negativeconformational energy; i.e., F=−E.

The “fitness contribution” of a protein residue refers to the level orextent f(i_(a)) to which the residue i_(a), having an identity (a),contributes to the total fitness of the protein. Thus, for example, ifchanging or mutating a particular amino acid residue will greatlydecrease the protein's fitness, that residue is said to have a highfitness contribution to the protein. By contrast, typically someresidues i_(a) in a protein may have a variety of possible identities(a) without affecting the protein's fitness. Such residues have a lowcontribution to the protein fitness.

“Dead-end elimination” (DEE) is a deterministic search algorithm thatseeks to systematically eliminate bad rotamers and combinations ofrotamers until a single solution remains. For example, amino acidresidues can be modeled as rotamers that interact with a fixed backbone.The theoretical basis for DEE provides that, if the DEE searchconverges, the solution is the global minimum energy conformation (GMEC)with no uncertainty (Desmet et al., 1992).

Dead end elimination is based on the following concept. Consider tworotamers, i_(r) and i_(t), at residue i, and the set of all otherrotamer configurations {S} at all residues excluding i (of which rotamerj_(s) is a member). If the pairwise energy contributed between i_(r) andj_(s) is higher than the pairwise energy between i_(t) and j_(s) for all{S}, then rotamer i_(t) cannot exist in the global minimum energyconformation, and can be eliminated. This notion is expressedmathematically by the inequality.

$\begin{matrix}{{{E\left( i_{r} \right)} + {\sum\limits_{j \neq i}^{N}{E\left( {i_{r},j_{s}} \right)}}} > {{E\left( i_{t} \right)} + {\sum\limits_{j \neq i}^{N}{{E\left( {i_{t},j_{s}} \right)}\left\{ S \right\}}}}} & \left( {{Equation}\mspace{14mu} A} \right)\end{matrix}$

If this expression is true, the single rotamer i_(r) can be eliminated(Desmet et al., 1992).

In this form, Equation A is not computationally tractable because, tomake an elimination, it is required that the entire sequence (rotamer)space be enumerated. To simplify the problem, bounds implied by EquationA can be utilized:

         (Equation  B)${{E\left( i_{r} \right)} + {\sum\limits_{j \neq i}^{N}{{\min (s)}{E\left( {i_{r},j_{s}} \right)}}}} > {{E\left( i_{t} \right)} + {\sum\limits_{j \neq i}^{N}{{\max (s)}{E\left( {i_{t},j_{s}} \right)}\left\{ S \right\}}}}$

Using an analogous argument, Equation B can be extended to theelimination of pairs of rotamers inconsistent with the GMEC. This isdone by determining that a pair of rotamers i_(r) at residue i and j_(s)at residue j, always contribute higher energies than rotamers i_(u) andj_(v) with all possible rotamer combinations {L}. Similar to Equation B,the strict bound of this statement is given by:

         (Equation  C)${\left( {i_{r},j_{s}} \right) + {\sum\limits_{{k \neq i},j}^{N}{{\min (t)}{ɛ\left( {i_{r},j_{s},k_{t}} \right)}}}} > {{ɛ\left( {i_{u},j_{v}} \right)} + {\sum\limits_{{k \neq i},j}^{N}{{\max (t)}{ɛ\left( {i_{u},j_{v},k_{i}} \right)}}}}$

where ε is the combined energies for rotamer pairs

ε(i _(r) j _(s))=E(i _(r))+E(j _(s))+E(i _(r) ,j _(s)   (Equation D),

and

ε(i _(r) j _(s) ,k _(t))=E(i _(r) ,k _(t))+E(j _(s) ,k _(t)   (EquationE).

This leads to the doubles elimination of the pair of rotamers i_(r) andj_(s), but does not eliminate the individual rotamers completely aseither could exist independently in the GMEC. The doubles eliminationstep reduces the number of possible pairs (reduces S) that need to beevaluated in the right-hand side of Equation 6, allowing more rotamersto be individually eliminated.

The singles and doubles criteria presented by Desmet et al. fail todiscover special conditions that lead to the determination of moredead-ending rotamers. For instance, it is possible that the energycontribution of rotamer i_(t) is always lower than i_(r)without themaximum of i_(t) being below the minimum of i_(r). A modification of thecriteria can be made that determines if the energy profiles of tworotamers cross. If they do not, the higher energy rotamer can bedetermined to be dead-ending. The doubles calculation may takesignificantly more computational time than the singles calculation. Toaccelerate the process, other computational methods have been developedto predict the doubles calculations that will be the most productive.See, for example, Gordon & Mayo, 1998, hereby incorporated by referenceit its entirety. These kinds of modifications, collectively referred toas fast doubles, significantly improved the speed and effectiveness ofDEE.

Several other modifications also enhance DEE. Rotamers from multipleresidues can be combined into so-called super-rotamers to prompt furthereliminations (Desmet et al., 1994; Goldstein, 1994).

For further discussion of these methods see, for example, Goldstein, R.F. (1994), Biophys. J. 66, 1335-1340; Desmet, J., De Maeyer, M., Hazes,B. & Lasters, I. (1992), Nature 356,539-542; Desmet, J., De Maeyer, M. &Lasters, I. (1994), In The Protein Folding Problem and TertiaryStructure Prediction (Jr., K. M. & Grand, S. L., eds.), pp. 307-337(Birkhauser, Boston); De Maeyer, M., Desmet, J. & Lasters, I. (1997),Folding & Design 2, 53-66, Gordon, D. B. & Mayo, S. L. (1998), J. ofComp. Chem. 19, 1505-1514; Pierce, N. A., Spriet, J. A., Desmet, J.,Mayo, S. L., (2000), J. of Comp. Chem. 21, 999-1009, all of which arehereby incorporated by reference in their entireties.

“Expression system” refers to herein a host cell and compatible vectorunder suitable conditions, e.g., for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells, Pseudomonas,or other bacterial cells and plasmid vectors, insect host cells such asSf9, Hi5 or S2 cells and Baculovirus vectors, Drosophila cells(Schneider cells) and expression systems, and mammalian host cells,including yeast and vectors, metazoan cells may also be used. Inaddition to E. coli, other specific host cells include yeast cells,Chinese hamster ovary (CHO) cells, fibroblast cells (BHK or Vero, forexample), stem cells (including embryonic stem cells), retinoblast cells(such as PerC.6 cells), hybridoma cells, neuronal cells, blood cells,bone marrow cells, liver cells, kidney cells, mammalian (includinghuman) embryonic cells of any origin, plasmacytoma cells (such as NS1cells), cell lines of any origin and hybrid-cross cells (including mixedmammalian cells, or cells from cross-species origin).

“Excipient,” generally refers to any agent, vehicle, carrier, binder,diluent, lubricant, surfactant, buffer, anti-aggregant, coloring,stabilizer, solubilizer, preservative, etc. that may be suitable for aparticular compound formulation. In certain aspects, the excipient mayimpart bulk to the formulation to make a tablet a practical size foradministration. In other aspects, the excipient may be an agent thatimparts cohesiveness to ensure the tablet remains intact aftercompression. In still other aspects, the excipient may be added tofacilitate breakup or disintegration of the solid dosage form afteradministration. In certain embodiments, the excipient may impartstability, solubility, or prevent aggregation of a liquid or lyophilizedformulation of a protein. Some examples of excipients include water,saline, celluloses, starches, clays, aligns, gums, talc, colloidalsilicon dioxide, lactose and other sugars, polymers, as well as variouscombinations of these or others. The excipient may comprise activematerials that do not impair the desired action, or with materials thatsupplement the desired action, or have another action. In addition,pharmaceutical or therapeutic carriers or vehicles may comprise anexcipient.

“Host cell” means any cell of any organism that is selected, modified,transformed, grown, used or manipulated in any way for the production ofa substance by the cell. A host cell may be auxotrophic, that is unableto synthesize or is deficient in at least one particular organiccompound required for its maintainence or growth and must obtain thecompound from another source, such as its environment or culture media.In addition, an auxotrophic host cell may have single, double, triple,quadruple, or more levels of auxotrophy such that it is unable tosynthesize one, two, three, four or more organic compounds necessary forits growth or maintainence, respectively. For example, a host cell maybe one that is manipulated to express a particular gene, a DNA or RNAsequence, a protein or an enzyme. Host cells may be cultured in vitro orin vivo in one or more cells in a non-human animal (e.g., a transgenicanimal or a transiently transfected animal).

The methods of the invention may include steps of comparing sequences toeach other, including a wild-type (also called “native”) sequence to oneor more mutants, or wild type sequences of the same gene from differentspecies or related genes of the same or different species. Suchcomparisons typically comprise alignments of gene or polypeptide(protein) sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, “—”, or “Δ”) in the polymer sequence notcontaining the inserted or deleted residue.

“Homologous”, in all of its grammatical forms and spelling variations,refers to the relationship between two molecules (e.g., proteins, tRNAs,nucleic acids) that possess a “common evolutionary origin”, includingproteins from superfamilies in the same species of organism, as well ashomologous proteins from different species of organism. Such proteins(and their encoding nucleic acids) have sequence and/or structuralhomology, as reflected by their sequence similarity, whether in terms ofpercent identity or by the presence of specific residues or motifs andconserved positions. Homologous molecules frequently also share similaror even identical functions.

The term “sequence similarity”, in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.However, in common usage and in the instant application, the term“homologous”, when modified with an adverb such as “highly”, may referto sequence similarity and may or may not relate to a commonevolutionary origin.

For example, any naturally occurring nucleic acid can be modified by anyavailable mutagenesis method to include one or more selector codon. Whenexpressed, this mutagenized nucleic acid encodes a polypeptidecomprising one or more non-natural amino acid. The mutation process can,of course, additionally alter one or more standard codon, therebychanging one or more standard amino acid in the resulting mutant proteinas well. Homology is generally inferred from sequence similarity betweentwo or more nucleic acids or proteins (or sequences thereof). Theprecise percentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establishhomology. If one or more particular amino acid or nucleic acid positionsexhibit higher levels of sequence similarity than others (among a groupof similar sequence(s) selected from different sources) then thepositions with higher sequence similarity are considered “highlyconserved.” Typically, but not always, the highly conserved regions of anucleic acid or amino acid sequence play an important role in thestructure and/or function of the molecule. Methods for determiningsequence similarity percentages (e.g., BLASTP and BLASTN using defaultparameters) are generally available.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (for example, see Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., hereby incorporated by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. For preliminary screening for homologous nucleicacids, low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used, (e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m) (e.g., 40% formamide, with 5× or 6×SSC). High stringencyhybridization conditions correspond to the highest T_(m) (e.g., 50%formamide, 5× or 6×SSC. SSC is a 0.15M NaCl, 0.015M Na-citrate).

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridizationmismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thus,the greater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.50-9.51, hereby incorporated by reference).For hybridization with shorter nucleic acids, i.e., oligonucleotides,the position of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8, hereby incorporated by reference). A minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 15 nucleotides; and more preferably the length is atleast about 20 nucleotides.

Unless specified, the term “standard hybridization conditions” refers toa T_(m) of about 55° C., and utilizes conditions as set forth above. Inat least one embodiment, the T_(m) is 60° C.; in at least oneembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Suitable hybridization conditions for oligonucleotides (e.g., foroligonucleotide probes or primers) are typically somewhat different thanfor full-length nucleic acids (e.g., full-length cDNA), because of theoligonucleotides' lower melting temperature. Because the meltingtemperature of oligonucleotides will depend on the length of theoligonucleotide sequences involved, suitable hybridization temperatureswill vary depending upon the oligonucleotide molecules used. Exemplarytemperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for17-base oligonucleotides), 55° C. (for 20-base oligonucleotides) and 60°C. (for 23-base oligonucleotides). Exemplary suitable hybridizationconditions for oligonucleotides include washing in 6×SSC/0.05% sodiumpyrophosphate, or other conditions that afford equivalent levels ofhybridization.

“Target molecule” used herein generally refers to a chemical orbiological entity which is capable of performing a chemical orbiological function or activity. “Target molecule” encompasses nucleicacids (DNA, RNA, etc.), proteins, polypeptides, peptides, biopolymers,carbohydrates, glycoproteins, glycolipids, lipids and the like and anycombination thereof. The methods of the present invention includemodifying a single target molecule or multiple target molecules. Ifmultiple target molecules are modified, they may be modifiedsequentially, simultaneously or otherwise. Furthermore, the chemical orbiological function or activity herein referred to may include functionsor activities similar to the corresponding native (wild type) targetmolecule(s) or it may include other functions, such as, for example,inhibiting the corresponding native (wild type) target molecule(s) oranother target molecule, increasing or decreasing the function of thecorresponding native (wild type) target molecule(s) or another targetmolecule, or otherwise affecting a chemical or biological mileu, cell,tissue, organ or system whether it be in vitro, in vivo, or ex vivo.

“Polypeptide,” “peptide” or “protein” are used interchangably todescribe a chain of amino acids that are linked together by chemicalbonds. A molecule, such as a protein or polypeptide, including anenzyme, may be a “native” or “wild-type”, meaning that it occurs innature; or it may be a “mutant,” “variant,” “derivative,” or“modification,” meaning that it has been made, altered, derived, or isin some way different or changed from a native molecule or from anothermutant. As used herein, “wild type” amino acid residue denotes thenative amino acid residue that naturally occurs in a particularmolecule, whereas “naturally occurring” amino acid residue may or maynot be a wild type amino acid residue. If used in context together, a“wild type” amino acid residue may be altered to another “naturallyoccurring” amino acid residue. In such a context, the phrase “naturallyoccurring” amino acid residue refers to any of the twenty naturallyoccurring amino acid residues, rather than any non-natural amino acid.Thus, a “wild type” amino acid residue located in a polypeptide, may bealtered to another “naturally occurring” amino acid residue differentthan the wild type amino acid residue, or to a “non-natural” amino acidresidue.

It is recognized in the art that polypeptide transcription reads thegene or polynucleotide from the 3′→5′ direction, resulting in apolypeptide generated in the 5′→3′ direction. As used herein, the firstposition refers to the amino acid (whether naturally occurring ornon-natural) at the 5′, (N), or amino terminus of the polypeptide, thesecond position refers to the amino acid at the second or penultimateposition of the polypeptide chain, the third position refers to the nextposition, and so on toward the 3′, (C), or carboxyl terminus. It isfurther understood that several “proof reading” functions occur bycellular machinery during polypeptide expression (transcription,translation, etc.) that may alter the gene or polynucleotide sequence.Thus, in one embodiment herein, the modified polynucleotide is altered(either by way of substitution or addition) to include one or morenon-natural amino acid codons. In certain embodiments, thepolynucleotide alterations occur such that when the host cell expressesthe polypeptide of interest, at least one non-natural amino acid residueretains the alterations of the gene or polynucleotide. In a preferredembodiment, the non-natural amino acid residue is at the first position(amino terminus) in the polypeptide and is retained during processing.In some embodiments, the efficiency of retention of the non-naturalamino acid residue at the first position of the N-terminal of thepolypeptide is increased by also altering the penultimate or secondposition of the polypeptide. The penultimate residue may be altered toanother naturally occurring amino acid or to a non-natural amino acid.In preferred embodiments, the side chains of the non-natural amino acidsincorporated into the modified polypeptide are unsaturated, therebyreducing side chain reactions or interactions with other amino acids inthe polypeptide. In some embodiments, the polypeptide is generatedwithout a host cell (in vitro, in silico, etc.) and non-natural aminoacid residues are incorporated during de novo protein synthesis.

A target molecule, such as a protein or polypeptide may also be referredto as “artificial,” which term includes a “mutant”, “variant”,“derivative” or “modification,” but further contains at least onenon-natural amino acid. As used herein, an “artificial polypeptide”includes, e.g., (a) a polynucleotide comprising a nucleotide sequenceencoding an artificial polypeptide of the invention; (b) apolynucleotide that is complementary to or that encodes a polynucleotidesequence of (a); (c) a nucleic acid that hybridizes to a polynucleotideof (a) or (b) under stringent conditions over substantially the entirelength of the nucleic acid; (d) a polynucleotide that is at least about95%, preferably at least about 98% identical to a polynucleotide of (a),(b), or (c); and (e) a polynucleotide comprising a conservativevariation of (a), (b), (c) or (d).

“Biopolymer” as used herein, refers to any natural or artificialbiological or chemical molecule, such as a protein, lipid orcarbohydrate that possesses additional polymeric characteristics ormodifications. A biopolymer may refer to a glycosylated or pegylated,myristylated, deamidated, or otherwise modified molecule for which apolymer has been joined, conjugated or intermixed.

“Rotamer” refers to a set of possible conformers for each amino acid oranalog side chain. See, for example Ponder, et al., Acad. Press Inc.(London) Ltd. pp. 775-791 (1987); Dunbrack, et al., Struc. Biol.1(5):334-340 (1994); Desmet, et al., Nature 356:539-542 (1992), all ofwhich are hereby incorporated by reference in their entireties.

A “rotamer library” is a collection of a set of possible/allowablerotametic conformations for a given set of amino acids or analogs. Thereare two general types of rotamer libraries: “backbone dependent” and“backbone independent.” A backbone dependent rotamer library allowsdifferent rotamers depending on the position of the residue in thebackbone; thus for example, certain leucine rotamers are allowed if theposition is within an α helix, and different leucine rotamers areallowed if the position is not in an α-helix. A backbone independentrotamer library utilizes all rotamers of an amino acid at everyposition. In general, a backbone independent library is preferred in theconsideration of core residues, since flexibility in the core isimportant. However, backbone independent libraries are computationallymore expensive, and thus for surface and boundary positions, a backbonedependent library is preferred. However, either type of library can beused at any position.

“Variable residue position” herein refers to an amino acid position ofthe protein to be designed that is not fixed in the design method as aspecific residue or rotamer, generally the wild-type residue or rotamer.It should be noted that even if a position is chosen as a variableposition, it is possible that the methods of the invention will optimizethe sequence in such a way as to select the wild type residue at thevariable position. This generally occurs more frequently for coreresidues, and less regularly for surface residues. In addition, it ispossible to fix residues as non-wild type amino acids as well.

“Fixed residue position” generally refers to the residue identified inthe three dimensional structure as being in a set conformation. In someembodiments, a fixed position is left in its original conformation(which may or may not correlate to a specific rotamer of the rotamerlibrary being used). Alternatively, residues may be fixed as a non-wildtype residue depending on design needs; for example, when knownsite-directed mutagenesis techniques have shown that a particularresidue is desirable, the residue may be fixed as a particular aminoacid. Residues which can be fixed include, but are not limited to,structurally or biologically functional residues.

In certain embodiments, a fixed position may be “floated”; the aminoacid or analog at that position is fixed, but different rotamers of thatamino acid or analog are tested. In this embodiment, the variableresidues may be at least one, or anywhere from 0.1% to 99.9% of thetotal number of residues. Thus, for example, it may be possible tochange only a few (or one) residues, or most of the residues, with allpossibilities in between.

As used herein, the term “mutant tRNA” or “mutant AARS” refers to a tRNAor AARs molecule that has reduced or no interaction or reaction withnative amino acids or endogenous unmodified transcriptional ortranslational machinery, and instead is able to interact or react withnon-natural amino acids and/or modified transcriptional or translationalmachinery, including other tRNA molecules and/or aminoacyl tRNAsynthetases. In certain embodiments, the mutant molecule reacts orinteracts with other mutant molecules and/or non-natural amino acids ata much greater effieciency than with naturally occurring amino acids ormolecules. In certain embodiments, the mutant molecule reacts orinteracts preferentially, and in certain embodiments, almostexclusively, with other mutant molecules and/or non-natural amino acids.For example, a mutant tRNA (M-tRNA) and/or a mutant aminoacyl tRNAsynthetase (M-RS) may be used with reduced efficiency (as compared towild-type or endogenous tRNA and/or AARS) by a system of interest (e.g.,a translational system, e.g., a cell). The M-tRNA and/or M-RS may alsobe referred to as “external mutant,” when the molecules are derived froma source other than the host cell in which they are being used forprotein translation. In other words, in certain embodiments the M-tRNAand/or M-RS molecules may be heterologous to the translation system.

As used herein, the term “external mutant” refers to a modified molecule(e.g., an external mutant tRNA and/or an external mutant aminoacyl tRNAsynthetase) that exhibits a reduced efficiency (as compared to wild-typeor endogenous) for aminoacylation with the corresponding wild type aminoacid. “External mutant” refers to the inability or reduced efficiency,e.g., less than 20% efficient, less than 10% efficient, less than 5%efficient, or, e.g., less than 1% efficient, of a tRNA and/or RS tofunction with the corresponding naturally occurring amino acid in thetranslation system of interest. For example, an external mutant RS in atranslation system of interest aminoacylates any endogenous tRNA of atranslation system of interest with the wild type amino acid at reducedor even zero efficiency, when compared to aminoacylation of anendogenous tRNA by the endogenous RS.

It should be noted, however, that an external mutant RS aminoacylates anendogenous tRNA with a replacement amino acid (whether naturallyoccurring or non-natural) with an increased efficiency compared with theability of the endogenous RS to aminoacylate an endogenous tRNA with areplacement amino acid. Likewise, an external mutant tRNA functions at ahigher efficiency toward the replacement amino acid codon (whether thereplacement amino acid comprises a non-natural or other naturallyoccurring amino acid) than toward the corresponding wild type aminoacid. Furthermore, an external mutant tRNA may function at an equal orhigher efficiency for a particular replacement amino acid codon (whetherthe replacement amino acid comprises a non-natural or other naturallyoccurring amino acid) than an endogenous tRNA.

A mutant tRNA and/or mutant AARS that reacts with a reduced efficiencyrefers to to the inability to react with, or reduced efficiency tointeract or react with, native amino acid residues, e.g., less than 20%efficient, less than 10% efficient, less than 5% efficient, or e.g.,less than 1% efficient.

In addition, “exogenous” tRNA and/or AARS molecules may be utilized incertain embodiments disclosed herein. In some embodiments, “exogenous”refers to a tRNA and/or AARS molecule that is derived from anotherorganism and may be wild type or mutant. Thus, an exogenous tRNA orexogenous AARS may also be an external mutant tRNA, or external mutantAARS, respectively.

“Wobble degenerate codon,” as used herein, refers to a codon encoding anaturally occurring amino acid, which codon, when present in mRNA, isrecognized by a natural tRNA anticodon through at least onenon-Watson-Crick, or wobble base-pairing (e.g., A-C or G-Ubase-pairing). Watson-Crick base-pairing refers to either the G-C or A-U(RNA or DNA/RNA hybrid) or A-T (DNA) base-pairing. When used in thecontext of mRNA codon—tRNA anticodon base-pairing, Watson-Crickbase-pairing means all codon-anticodon base-pairings are mediatedthrough either G-C or A-U. “Wobble decoding,” then, generally refers tothe ability of a particular tRNA to read through non-Watson-Crick basepairing.

“Bias codon,” as used herein, refers to a degenerate codon that encodesa naturally occurring amino acid, which codon is one that is used by atRNA (“bias codon tRNA”) which bias codon tRNA is present in aparticular host cell at a lower concentration relative to other tRNAmolecules used for the same naturally occurring amino acid. In certainembodiments, the lower frequency of the bias codon tRNA may be theresult of modification of the host cell in order to reduce the level oravailability of the bias codon tRNA in the cell. This may beaccomplished, for example, by way of deletion or inactivation of thespecific bias codon tRNA gene(s) from the genome of the host cell. Incertain embodiments, the bias codon tRNA is present at a frequency ofless than about 25%, less than about 15%, less than about 10%, less thanabout 8%, less than about 5%, less than about 4%, less than about 3%,less than about 2%, less than about 1%, less than about 0.5%, less thanabout 0.4%, less than about 0.3%, less than about 0.2%, less than about0.1%, less than about 0.05%, less than about 0.01%, or less than thefrequency of the most common tRNA that is utilized for the same codon inthe translation system.

“Sixth box codon,” as used herein, refers to any one of six codons thatencode the same naturally occurring or non-natural amino acid (includingbut not limited to arginine, leucine, or serine). For embodiments inwhich a sixth box codon specifies a non-natural amino acid, the sixthbox codon is not recognized by at least one tRNA that decodes the otherfive codons encoding the same amino acid residue. This lack ofrecognition by the sixth box codon tRNA allows the sixth box codon tospecify a position for incorporation of the non-natural amino acid thatcorresponds to the naturally occurring amino acid. In this case, thenaturally occurring amino acid is able to incorporate in the targetmolecule at other positions in the same target molecule sicne it isencoded by codons that are not recognized by the sixth box codon tRNA.Examples of sixth box codons include the CGA, AGG and AGA codons forarginine, or CTA for leucine. Other degenerate codons are listed in thetables entitled, “The Genetic Code, ” and “The Degenerate Codons for E.coli” inter alia herein.

Similar to the sixth box codon is a two or four box degenerate codon forwhich there is a tRNA that will not wobble decode another of thedegenerate codons for the same amino acid.

In still other embodiments of the present invention, artificialanticodons may be created to form Watson-Crick base pairing with wobblecodons.

One of skill in the art would understand that an anticodon generallyrefers to the nucleotide sequence (typically 3 nucleotides in length butmay be 2, 3, 4, 5 nucleotides in length, or other size) that iscomplementary (either by Watson-Crick base pairing or wobble pairing) tothe nucleotide codon present on the corresponding messenger RNAmolecule. During protein translation, the anticodon on the tRNA moleculeis matched to a specific amino acid that is then covalently attached tothe tRNA. In certain embodiments, the anticodon matches a correspondingcodon that comprises a stop codon, including a nonsense codon ormissense codon. In this way, altering the anticodon may allow forspecific incorporation of a non-natural amino acid in to a targetmolecule. An artificial anticodon, then, may be any codon that has beenaltered (at the nucleic acid level or amino acid level) to allow forincorporation of an amino acid (whether naturally occurring amino acidor non-natural amino acid) into a target molecule.

“Borrowed codon,” as used herein, generally refers to a codon for afirst naturally occurring amino acid or non-natural amino acid that isrecognized by an endogenous or exogenous tRNA or M-tRNA that is capableof being aminoacylated by the corresponding AARS of the first aminoacid, but which is actually aminoacylated by a chimeric M-RS. A“chimeric M-RS” refers to an AARS which contains the structures from theAARS of the first amino acid that bind to tRNA identity elements,combined with the amino acid binding domain from an AARS for a secondamino acid such that the second amino acid is incorporated in the targetmolecule at the borrowed codon site. In certain embodiments, thechimeric M-RS may be modified to bind a non-natural amino acid, suchthat the non-natural amino acid is incorporated at the borrowed codonsite. The borrowed codon may include codons that may be decoded bynaturally occurring or artificial anticodons. In certain embodimentswherein an artificial anticodon is utilized, the anticodon may becreated to form Watson-Crick base pairing with wobble codons for aparticular amino acid.

The term “preferentially aminoacylates” refers to an efficiency, e.g.,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about75%, about 85%, about 90%, about 95%, about 99% or more efficient. Theefficiency may be measured by which a modified or external mutantaminoacyl tRNA synthetase aminoacylates a tRNA with a replacement aminoacid, whether an unnatural amino acid or another naturally occurringamino acid when compared to the corresponding natural amino acidassigned to the particular tRNA, AARS, or both.

The term “preferentially aminoacylates” further may refer to theefficiency of the modified or external mutant aminoacyl tRNA synthetaseto aminoacylate or charge a tRNA with any amino acid other than thecorresponding natural amino acid assigned to the particular tRNA, AARS,or both. The term “preferentially aminoacylates” further may refer tothe efficiency of the modified or external mutant aminoacyl tRNAsynthetase to aminoacylate a tRNA with a non-natural amino acid comparedwith the non-modified or naturally occurring AARS. In certainembodiments, “preferentially aminoacylates” further relates to theefficiency as measured by the kinetics in which a modified or externalmutant AARS aminoacylates a tRNA with another amino acid (as describedby Km, kcat, kcat/Km, or ATP-PPi exchange rate).

It should be noted that the efficiency of aminoacylation of the tRNA bythe AARS may be correlated to the efficiency of specificity, or fidelityof incorporation of the non-natural amino acid in the target polypeptideor protein. This is due to the function of the protein synthesismachinery in that once a tRNA is aminoacylated with an amino acid(whether the wild type amino acid, or a non-natural amino acid), thecharged tRNA is released from the AARS enzyme and the amino acid isincorporated into the target polypeptide. When the proofreading abilityof the AARS is altered, the enzyme will allow the replacement amino acidto charge the tRNA and be released for incorporation into the targetprotein. Thus, the efficiency of aminoacylation by the AARS directlycorrelates to the fidelity or specificity of incorporation of thenon-natural amino acid into the target polypeptide.

The replacement (whether non-natural or naturally occurring) amino acidis then incorporated into a growing polypeptide chain with highfidelity, e.g., at greater than about 20%, 30%, 40%, 50%, 60%, 75%, 80%,90%, 95%, or greater than about 99% efficiency for a particular codon.

The modified AARS may be altered such that the binding efficiency to thenon-natural amino acid, or another selected naturally occurring aminoacid, is greater than the binding efficiency of the modified AARS to thecorresponding naturally occurring amino acid. In this way, a modifiedAARS may preferentially bind a non-natural amino acid in order to chargea tRNA even in the presence of the naturally occurring amino acid thatcorresponds to the AARS in its unmodified state. This “reprogramming” ofan aminoacyl tRNA synthetase allows for incorporation of a non-naturalamino acid into a polypeptide with lower levels of mis-incorporation ofother amino acids into the desired site.

The “reprogramming” further may allow for use of the modified orexternal mutant synthetase with high levels of incorporation in standardhost cells, without the need for auxotrophic host cells, and with orwithout depleting the media of the corresponding naturally occurringamino acid. Thus, while certain embodiments disclosed herein may bepracticed by using an auxotrophic host cell, certain other embodimentsmay be practiced without using an auxotrophic host cell. In the event ofnot using an auxotrophic host cell to practice certain embodiments,another host cell may be used, cellular components may be used, or anentirely cell-free system may be used.

The term “complementary” refers to components of an external mutantpair, the external mutant tRNA and external mutant synthetase that canfunction together, e.g., the external mutant synthetase aminoacylatesthe external mutant tRNA.

The term “derived from” refers to a component that is isolated from anorganism or isolated and modified, or generated, e.g., chemicallysynthesized, using information of the component from the organism.

The term “translation system” refers to the components necessary toincorporate a naturally occurring or non-natural amino acid into agrowing polypeptide chain (protein). For example, components can includeribosomes, tRNA(s), synthetase(s), mRNA and the like. The componentsdisclosed herein can be added to a translation system, in vivo or invitro. An in vivo translation system may be a cell (eukaryotic orprokaryotic cell). An in vitro translation system may be a cell-freesystem, such as a reconstituted one with components from differentorganisms (purified or recombinantly produced). In certain embodiments,the translation system does not comprise a cell. In certain embodiments,the translation system does not comprise an auxotrophic cell. If thetranslation system does not comprise an auxotrophic cell, it maycomprise another cell or cellular components.

The term “inactive RS” refers to a synthetase that has been mutated sothat it no longer can aminoacylate its cognate tRNA with any amino acid,whether naturally occurring or non-natural. The term “modified RS”refers to a synthetase that has been mutated such that it no longer canaminoacylate its cognate tRNA with the corresponding naturally occurringamino acid, but may be able to aminoacylate its cognate tRNA withanother amino acid, preferably a non-natural amino acid.

The term “not efficiently recognized” refers to an efficiency, e.g.,less than about 10%, less than about 5%, or less than about 1%, at whicha RS from one organism aminoacylates an external mutant tRNA. In certainembodiments, the RS may be from the same or a different organism thanthe external mutant tRNA. In some embodiments, the RS has been modifiedto aminoacylate a tRNA with a particular amino acid, preferably anon-natural amino acid.

The term “selection agent” refers to an agent that when present allowsfor a selection of certain components from a population, e.g., anantibiotic, wavelength of light, an antibody, a nutrient or the like.The selection agent can be varied, e.g., such as concentration,intensity, etc.

The term “positive selection marker” refers to a marker than whenpresent, e.g., expressed, activated or the like, results inidentification of an organism with the positive selection marker fromthose without the positive selection marker.

The term “negative selection marker” refers to a marker than whenpresent, e.g., expressed, activated or the like, allows identificationof an organism that does not possess the desired property (e.g., ascompared to an organism which does possess the desired property).

The term “reporter” refers to a component that can be used to selectcomponents described in the present invention. For example, a reportercan include a green fluorescent protein, a firefly luciferase protein,or genes such as β-gal/lacZ (β-galactosidase), Adh (alcoholdehydrogenase) or the like.

The term “eukaryote” refers to organisms belonging to the phylogeneticdomain Eucarya such as animals (e.g., mammals, insects, reptiles, birds,etc.), ciliates, plants, fungi (e.g., yeasts, etc.), flagellates,microsporidia, protists, etc. Additionally, the term “prokaryote” refersto non-eukaryotic organisms belonging to the Eubacteria (e.g.,Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g.,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, A. fulgidus, P. firiosus, P. horikoshii, A. pernix, etc.)phylogenetic domains.

The term “pharmaceutical” or “pharmaceutical drug,” as used hereinrefers to any pharmacological, therapeutic or active biological agentthat may be administered to a subject. In certain embodiments thesubject is an animal, including a vertebrate, and preferably a mammal,most preferably a human. In certain embodiments the animal is avertebrate. In certain embodiments the animal is a mammal. In certainembodiments the animal is a human.

The term “pharmaceutically acceptable carrier,” as used herein, refersgenerally to any material that may accompany the pharmaceutical drug butwhich does not interfere with the activity of the pharmaceutical drugand which does not cause an adverse reaction with the subject's immunesystem.

As used herein, the term “administering,” refers to any mode oftransferring, delivering, introducing or transporting a pharmaceuticaldrug or other agent to a subject. Such modes include oraladministration, topical contact, intravenous, intraperitoneal,intramuscular, intralesional, intranasal, subcutaneous or intrathecaladministration. Also contemplated by the present invention isutilization of a device or instrument in administering an agent. Suchdevice may utilize active or passive transport and may be slow-releaseor fast-release delivery device.

As used herein, the term “saccharide moiety” refers to natural andnon-natural sugar moieties (i.e., a non-naturally occurring sugarmoiety, e.g., a sugar moiety that is modified, e.g., at one or morehydroxyl or amino positions, e.g., dehydroxylated, deaminated,esterified, etc., e.g., 2-deoxyGal is an example of an non-natural sugarmoiety).

The term “carbohydrate” has the general formula (CH₂O)_(n), andincludes, but is not limited to, e.g., monosaccharides, disaccharides,oligosaccharides and polysaccharides. Oligosaccharides are chainscomposed of saccharide units, which are alternatively known as sugars.Saccharide units can be arranged in any order and the linkage betweentwo saccharide units can occur in any of approximately ten differentways. The following abbreviations are used herein: Ara=arabinosyl;Fru=fructosyl; Fuc=fucosyl; Gal=galactosyl;GaINAc=N-acetylgalactosaminyl; Glc=glucosyl;GlcNAc=N-acetylglucosaminyl; Man=mannosyl; and NeuAc=sialyl (typicallyN-acetylneuraminyl).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right. All oligosaccharides describedherein are described with the name or abbreviation for the non-reducingsaccharide (e.g., Gal), followed by the configuration of the glycosidicbond (α or β), the ring bond, the ring position of the reducingsaccharide involved in the bond, and then the name or abbreviation ofthe reducing saccharide (e.g., GIcNAc). The linkage between two sugarsmay be expressed, for example, as 2,3; 2→3; 2-3; or (2,3). Natural andnon-natural linkages (e.g., 1-2; 1-3; 1-4; 1-6; 2-3; 2-4; 2-6; etc.)between two sugars are included in the invention. Each saccharide is apyranose.

The term “sialic acid” (abbreviated “Sia”) refers to any member of afamily of nine-carbon carboxylated sugars. The most common member of thesialic acid family is N-acetyl-neuraminic acid(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid) (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al., J. Biol.Chem. 261: 11550-11557, 1986; Kanamori et al., J. Biol. Chem. 265:21811-21819, 1990). Also included are 9-substituted sialic acids such asa 9-O-C1-C6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40, 1992;Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is described in, for example,international application WO 92/16640 (entire contents incorporatedherein by reference).

Donor substrates for glycosyl transferases are activated nucleotidesugars. Such activated sugars generally consist of uridine and guanosinediphosphate, and cytidine monophosphate, derivatives of the sugars inwhich the nucleoside diphosphate or monophosphate serves as a leavinggroup. Bacterial, plant, and fungal systems can sometimes use otheractivated nucleotide sugars.

The Genetic Code and the Degenerate Codons

The standard genetic code most cells use is listed below.

The Genetic Code Middle First U C A G Last Phe Ser Tyr Cys U Phe Ser TyrCys C U Leu Ser Stop (Ochre) Stop (Umber) A Leu Ser Stop (Amber) Trp GLeu Pro His Arg U Leu Pro His Arg C C Leu Pro Gln Arg A Leu Pro Gln ArgG Ile Thr Asn Ser U A Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr LysArg G Val Ala Asp Gly U G Val Ala Asp Gly C Val Ala Glu Gly A Val AlaGlu Gly G

The genetic code is degenerate, in that the protein biosyntheticmachinery utilizes 61 mRNA sense codons to direct the templatedpolymerization of the 20 natural amino acid monomers. (See, for example,Crick et al., Nature 192: 1227, 1961, hereby incorporated by reference).Two amino acids (methionine and tryptophan), are encoded by unique mRNAtriplets.

The standard genetic code applies to most, but not all, cases.Exceptions have been found in the mitochondrial DNA of many organismsand in the nuclear DNA of a few lower organisms. Some examples are givenin the following table.

Examples of Non-Standard Genetic Codes

Mitochondria Vertebrates UGA→ Trp; AGA, AGG → STOP Invertebrates UGA→Trp; AGA, AGG → Ser Yeasts UGA→ Trp; CUN → Thr Protista UGA→ Trp;Nucleus Bacteria GUG, UUG, AUU, CUG → initiation Yeasts CUG → SerCiliates UAA, UAG → Gln *Plant cells use the standard genetic code inboth mitochondria and the nucleus.

The NCBI (National Center for Biotechnology Information) maintains adetailed list of the standard genetic code, and genetic codes used invarious organisms, including the vertebrate mitochondrial code; theyeast mitochondrial code; the mold, protozoan, and coelenteratemitochondrial code and the mycoplasma/spiroplasma code; the invertebratemitochondrial code; the ciliate, dasycladacean and hexamita nuclearcode; the echinoderm and flatworm mitochondrial code; the euplotidnuclear code; the bacterial and plant plastid code; the alternativeyeast nuclear code; the ascidian mitochondrial code; the alternativeflatworm mitochondrial code; blepharisma nuclear code; chlorophyceanmitochondrial code; trematode mitochondrial code; scenedesmus obliquusmitochondrial code; thraustochytrium mitochondrial code (allincorporated herein by reference). These are primarily based on thereviews by Osawa et al., Microbiol. Rev. 56: 229-264, 1992, and Jukesand Osawa, Comp. Biochem. Physiol. 106B: 489-494, 1993, all herebyincorporated by reference in their entireties.

Degenerate Codon Selection

As described above, all amino acids, with the exception of methionineand tryptophan are encoded by more than one codon. According to themethods of the invention, a codon that is normally used to encode anatural amino acid is reprogrammed to encode an amino acid analog. Anamino acid analog can be a naturally occurring or canonical amino acidanalog. In a preferred embodiment, the amino acid analog is not acanonically encoded amino acid.

The following table lists some of the known anti-codon sequences for E.coli. In general, for any organism, tRNA anticodon sequence can beroutinely determined using art-recognized technologies. For example, anytRNA gene can be amplified by, for example, PCR. Sequencing can beperformed to determine the exact sequences of the anti-codon loop.Alternatively, biochemical binding assay may be used to determine thebinding affinity of a purified tRNA to one of the 2-6 possible codons.The codon that binds the tRNA with the highest specificity/affinitypresumably has pure Watson-Crick match at all three codon positions,thus determining the sequence of the anti-codon loop.

In general, the wobble base in the anti-codon loop tends to be G or U(rather than A or C), but is not limited to such.

The Degenerate Codons for E. coli Base- A- paring A- mino Anti- at3^(rd) mino Anti- Base- Acid codon base Codon Acid codon paring CodonAla GGC W/C¹ GCC His GUG W/C CAC Wobble² GCU Wobble CAU UGC W/C GCA IleGAU W/C AUC Wobble GCG Wobble AUU, AUA Asp GUC W/C GAC Leu GAG W/C CUC,CUA, CUG, UUC, UUG Wobble GAU Wobble CUU Asn GUU W/C AAC Lys UUU W/C AAAWobble AAU Wobble AAG Cys GCA W/C UGC Phe GAA W/C UUC Wobble UGU WobbleUUU Glu UUC W/C GAA Ser GGA W/C UUC, AGU Wobble GAG Wobble UCU, AGC,UCA, UCG Gly GCC W/C GGC, Tyr GUA W/C UAC GGA, GGG Wobble GGU Wobble UAUMet W/C AUG Thr W/C ACC, ACA, ACG Gln W/C CAA, Wobble ACU CAG Arg W/CAGA, Pro W/C CCC, AGG, CCA, CGU, CCG CGG Wobble CGC, Trp Wobble CCU CGAW/C UGG STOP W/C UGA, Val W/C GUC, UAA GUA Wobble UAG Wobble GUU, GUG¹Watson-Crick base pairing ²Wobble base pairing

When the cell has a single tRNA that recognizes a codon through aperfect complementary interaction between the anticodon of the tRNA andone codon, and recognizes a second, degenerate codon through a wobble orother non-standard base pairing interaction, a new tRNA can beconstructed having an anticodon sequence that is perfectly complementaryto the degenerate codon.

When the cell has multiple tRNA molecules for a particular amino acid,and one tRNA has an anticodon sequence that is perfectly complementaryto the degenerate codon selected, the gene encoding the tRNA can bedisabled through any means available to one of skill in the art. Suchexemplary means include chemical mutagenesis, DNA shuffling or geneshuffling (including genetic recombination), randomized geneticmutagenesis, site-directed mutagenesis or deletion of either the gene orthe promoter sequence of the gene. Expression of the gene also can bedisabled through any antisense or RNA interference techniques.

The deletion or disablement of a tRNA will result in the disablement ofthe corresponding codon which may be fatal to the host cell. In order torescue the host cell, such tRNA disablement may be accompanied by theintroduction of a tRNA gene whose expression is regulated. Theregulation of the tRNA expression may be accomplished by using arepressible promoter (such as copper ion inducible and repressiblepromoter systems in yeast). See, for example, Meth. Enzymol. 306:145-153 (1999), hereby incorporated by reference in its entirety. Theregulated tRNA will function to support host cell growth before the geneof interest in induced, and the tRNA will be repressed prior to or whenthe gene of interest is induced in the presence of the non-natural aminoacid. The non-natural amino acid is incorporated by an exogenous tRNA orM-tRNA capable of decoding the same codon, but which only functions withits cognate M-RS and in the presence of the non-natural amino acid.

Alternatively, the disablement of the tRNA may be accomplished with aninterfering RNA (iRNA), or antisense, expression of both of which may beregulated. In this case, the iRNA or antisense expression may be inducedby the same agent (e.g., IPTG) as well as for inducing expression of thetarget molecule. The addition of the non-natural amino acid will enablethe exogenous or M-tRNA and M-RS to use the same codon disabled bydeletion or disablement of the endogenous tRNA.

Unnatural or Non-Natural Amino Acids

The first step in the protein engineering process is usually to select aset of non-natural amino acids that have the desired chemicalproperties. The selection of non-natural amino acids depends onpre-determined chemical properties and the modifications one would liketo make in the target molecule or target protein. Non-natural aminoacids, once selected, can either be purchased from vendors, orchemically synthesized. Any number of non-natural amino acids may beincorporated into the target molecule and may vary according to thenumber of desired chemical moieties that are to be attached. Thechemical moieties may be attached to all or only some of the non-naturalamino acids. Further, the same or different non-natural amino acids maybe incorporated into the molecule, depending on the desired outcome. Incertain embodiments, at least two different non-natural amino acids areincorporated into the molecule and one chemical moiety, such as PEG, isattached to one of the non-natural amino acid residues, while anotherchemical moiety, such as a cytotoxic agent, is attached to the othernon-natural amino acid.

A wide variety of non-natural amino acids can be used in the methods ofthe invention. Typically, the non-natural amino acids of the inventionare selected or designed to provide additional characteristicsunavailable in the twenty natural amino acids. For example, non-naturalamino acids are optionally designed or selected to modify the biologicalproperties of a molecule, including a protein, e.g., into which they areincorporated. For example, the following properties are optionallymodified by inclusion of an non-natural amino acid into a molecule, suchas a protein: toxicity, biodistribution, solubility, stability, e.g.,thermal, hydrolytic, oxidative, resistance to enzymatic degradation, andthe like, facility of purification and processing, structuralproperties, spectroscopic properties, chemical and/or photochemicalproperties, catalytic activity, ability to function as a vaccine, redoxpotential, half-life, ability to react with other molecules, e.g.,covalently or noncovalently, and the like.

As used herein an “non-natural amino acid” refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand the following twenty genetically encoded alpha-amino acids: alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.The generic structure of an alpha-amino acid is illustrated by FormulaI:

An non-natural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., any biochemistry text such asBiochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York,for structures of the twenty natural amino acids. Note that thenon-natural amino acids disclosed herein may be naturally occurringcompounds other than the twenty alpha-amino acids above.

Because the non-natural amino acids disclosed herein typically differfrom the natural amino acids in side chain only, the non-natural aminoacids form amide bonds with other amino acids, e.g., natural ornon-natural, in the same manner in which they are formed in naturallyoccurring proteins. However, the non-natural amino acids have side chaingroups that distinguish them from the natural amino acids. For example,R in Formula I optionally comprises an alkyl-, aryl-, aryl halide, vinylhalide, alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide,acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide,alkenyl, alkynyl, ether, thioether, epoxide, sulfone, boronic acid,boronate ester, borane, phenylboronic acid, thiol, seleno-, sulfonyl-,borate, boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl,naphthyl, benzophenone, a constrained ring such as a cyclooctyne,thioester, enone, imine, aldehyde, ester, thioacid, hydroxylamine,amino, carboxylic acid, alpha-keto carboxylic acid, alpha or betaunsaturated acids and amides, glyoxyl amide, or organosilane group, orthe like or any combination thereof.

Specific examples of unnatural amino acids include, but are not limitedto, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GIcNAcβ-serine, β-O-GIcNAc-L-serine, atri-O-acetyl-GaINAc-α-threonine, an α-GaINAc-L-threonine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, those listedbelow, or elsewhere herein, and the like.

Aryl substitutions may occur at various positions, e.g. ortho, meta,para, and with one or more functional groups placed on the aryl ring.Other non-natural amino acids of interest include, but are not limitedto, amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, dye-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids withaltered hydrophilicity, hydrophobocity, polarity, or ability to hydrogenbond, amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, amino acidscomprising biotin or a biotin analogue, glycosylated amino acids such asa sugar substituted serine, other carbohydrate modified amino acids,keto containing amino acids, amino acids comprising polyethylene glycolor a polyether, a polyalcohol, or a polysaccharide, amino acids that canundergo metathesis, amino acids that can undergo cycloadditions, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, e.g., polyethers or long chain hydrocarbons, e.g.,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, amino acids containing a drug moiety, and aminoacids comprising one or more toxic moieties.

In addition to non-natural amino acids that contain novel side chains,non-natural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH₂, SH, NH₂O—, NH—R′, R′NH—, R′S—, orS—R′—; X and Y, which may be the same or different, typically compriseS, N, or O, and R and R′, which are optionally the same or different,are typically selected from the same list of constituents for the Rgroup described above for the non-natural amino acids having Formula Ias well as hydrogen or (CH₂)_(x) or the natural amino acid side chains.For example, non-natural amino acids disclosed herein optionallycomprise substitutions in the amino or carboxyl group as illustrated byFormulas II and III. Non-natural amino acids of this type include, butare not limited to, α-hydroxy acids, α-thioacidsα-aminothiocarboxylates, or α-α-disubstituted amino acids, with sidechains corresponding, e.g., to the twenty natural amino acids or tonon-natural side chains. They also include but are not limited toβ-amino acids or γ-amino acids, such as substituted β-alanine andγ-amino butyric acid. In addition, substitutions or modifications at theα-carbon optionally include L or D isomers, such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogs as well as 3-, 4-, 6-, 7-, 8-, and 9- membered ring prolineanalogs. Some non-natural amino acids, such as aryl halides(p-bromo-phenylalanine, p-iodophenylalanine, provide versatile palladiumcatalyzed cross-coupling reactions with ethyne or acetylene reactionsthat allow for formation of carbon-carbon, carbon-nitrogen andcarbon-oxygen bonds between aryl halides and a wide variety of couplingpartners.

For example, many non-natural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like.Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C6-C20 straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs include, but are notlimited to, α-hydroxy derivatives, β-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Exemplaryphenylalanine analogs include, but are not limited to, meta-substitutedphenylalanines, wherein the substituent comprises a hydroxy group, amethoxy group, a methyl group, an allyl group, an acetyl group, or thelike.

Specific examples of non-natural amino acids include, but are notlimited to, o, m and/or p forms of amino acids or amino acid analogs(non-natural amino acids), including homoallylglycine, cis- ortrans-crotylglycine, 6,6,6-trifluoro-2-aminohexanoic acid,2-aminopheptanoic acid, norvaline, norleucine, O-methyl-L-tyrosine, o-,m-, or p-methyl-phenylalanine, O-4-allyl-L-tyrosine, a4-propyl-L-tyrosine, a tri-O-acetyl-GIcNAcβ-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azidophenylalanine, a p-acyl-L-phenylalanine, ap-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, o-, m-, orp-bromophenylalanine, 2-, 3-, or 4-pyridylalanine, p-idiophenylalanine,diaminobutyric acid, aminobutyric acid, benzofuranylalanine,3-bromo-tyrosine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine,3-(5-bromonindolyl)alanine, p-chlorophenylalanine,p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine,m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1 H-pyrol-3-yl)propanoic acid, azidonorleucine,azidohomoalanine, p-acetylphenylalanine, p-amino-L-phenylalanine,homoproparglyglycine, p-ethyl-phenylalanine, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, isopropyl-L-phenylalanine, an3-(2-naphthyl)alanine, 3-(1-naphthyl)alanine, 3-idio-tyrosine,O-propargyl-tyrosine, homoglutamine, an O-4-allyl-L-tyrosine, a4-propyl-L-tyrosine, a 3-nitro-L-tyrosine, atri-O-acetyl-GIcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-acetyl-L-phenylalanine, anm-acetyl-L-phenylalanine, selenomethionine, telluromethionine,selenocysteine, an alkyne phenylalanine, an O-allyl-L-tyrosine, anO-(2-propynyl)-L-tyrosine, a p-ethylthiocarbonyl-L-phenylalanine, ap-(3-oxobutanoyl)-L-phenylalanine, a p-benzoyl-L-phenylalanine, anL-phosphoserine, a phosphonoserine, a phosphonotyrosine,homoproparglyglycine, azidohomoalanine, a p-iodo-phenylalanine, ap-bromo-L-phenylalanine, dihydroxy-phenylalanine,dihydroxyl-L-phenylalanine, a p-nitro-L-phenylalanine, anm-methoxy-L-phenylalanine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, trifluoroleucine, norleucine, 4-, 5-, or 6-fluoro-tryptophan, 4-aminotryptophan, 5-hydroxytryptophan, biocytin,aminooxyacetic acid, m-hydroxyphenylalanine, m-allyl phenylalanine,m-methoxyphenylalanine group, β-GlcNAc-serine, α-GaINAc-threonine,p-acetoacetylphenylalanine, para-halo-phenylalanine, seleno-methionine,ethionine, S-nitroso-homocysteine, thia-proline, 3-thienyl-alanine,homo-allyl-glycine, trifluoroisoleucine, trans andcis-2-amino-4-hexenoic acid, 2-butynyl-glycine, allyl-glycine,para-azido-phenylalanine, para-cyano-phenylalanine,para-ethynyl-phenylalanine, hexafluoroleucine, 1,2,4-triazole-3-alanine,2-fluoro-histidine, L-methyl histidine, 3-methyl-L-histidine,β-2-thienyl-L-alanine, β-(2-thiazolyl)-DL-alanine, homoproparglyglycine(HPG) and azidohomoalanine (AHA) and the like. The structures of avariety of non-limiting non-natural amino acids are provided in thefigures, e.g., FIGS. 29, 30, and 31 of US 2003/0108885 A1, the entirecontent of which is incorporated herein by reference.

Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C6-C20 straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs of the inventioninclude, but are not limited to, α-hydroxy derivatives, β-substitutedderivatives, cyclic derivatives, and amide substituted glutaminederivatives. Example phenylalanine analogs include, but are not limitedto, meta-substituted phenylalanines, wherein the substituent comprises ahydroxy group, a methoxy group, a methyl group, an allyl group, anacetyl group, or the like.

Additionally, other examples optionally include (but are not limited to)an non-natural analog of a tyrosine amino acid; an non-natural analog ofa glutamine amino acid; an non-natural analog of a phenylalanine aminoacid; an non-natural analog of a serine amino acid; an non-naturalanalog of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano,halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol,sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or any combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; an amino acid with a novel functional group; anamino acid that covalently or noncovalently interacts with anothermolecule; a metal binding amino acid; a metal-containing amino acid; aradioactive amino acid; a photocaged amino acid; a photoisomerizableamino acid; a biotin or biotin-analog containing amino acid; aglycosylated or carbohydrate modified amino acid; a keto containingamino acid; an amino acid comprising polyethylene glycol; an amino acidcomprising polyether; a heavy atom substituted amino acid; a chemicallycleavable or photocleavable amino acid; an amino acid with an elongatedside chain; an amino acid containing a toxic group; a sugar substitutedamino acid, e.g., a sugar substituted serine or the like; acarbon-linked sugar-containing amino acid; a redox-active amino acid; anα-hydroxy containing acid; an amino thio acid containing amino acid; anα,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid.

Typically, the non-natural amino acids utilized herein for certainembodiments may be selected or designed to provide additionalcharacteristics unavailable in the twenty natural amino acids. Forexample, non-natural amino acid are optionally designed or selected tomodify the biological properties of a protein, e.g., into which they areincorporated. For example, the following properties are optionallymodified by inclusion of an non-natural amino acid into a protein:toxicity, biodistribution, solubility, stability, e.g., thermal,hydrolytic, oxidative, resistance to enzymatic degradation, and thelike, facility of purification and processing, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic activity, redox potential, half-life, ability to react withother molecules, e.g., covalently or noncovalently, and the like.

Other examples of amino acid analogs optionally include (but are notlimited to) an non-natural analog of a tyrosine amino acid; annon-natural analog of a glutamine amino acid; an non-natural analog of aphenylalanine amino acid; an non-natural analog of a serine amino acid;an non-natural analog of a threonine amino acid; an alkyl, aryl, acyl,azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl,ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate,phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde,hydroxylamine, keto, or amino substituted amino acid, or any combinationthereof; an amino acid with a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; an amino acid with anovel functional group; an amino acid that covalently or noncovalentlyinteracts with another molecule; a metal binding amino acid; ametal-containing amino acid; a radioactive amino acid; a photocagedamino acid; a photoisomerizable amino acid; a biotin or biotin-analoguecontaining amino acid; a glycosylated or carbohydrate modified aminoacid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol; an amino acid comprising polyether; a heavy atomsubstituted amino acid; a chemically cleavable or photocleavable aminoacid; an amino acid with an elongated side chain; an amino acidcontaining a toxic group; a sugar substituted amino acid, e.g., a sugarsubstituted serine or the like; a carbon-linked sugar-containing aminoacid; a redox-active amino acid; an α-hydroxy containing acid; an aminothio acid containing amino acid; an α,α disubstituted amino acid; aβ-amino acid; and a cyclic amino acid other than proline.

Non-natural amino acids suitable for use in the methods of the inventionalso include those that have a saccharide moiety attached to the aminoacid side chain. In one embodiment, an non-natural amino acid with asaccharide moiety includes a serine or threonine amino acid with a Man,GaINAc, Glc, Fuc, or Gal moiety. Examples of non-natural amino acidsthat include a saccharide moiety include, but are not limited to, e.g.,a tri-O-acetyl-GIcNAcβ-serine, a β-O-GlcNAc-L-serine, atri-O-acetyl-GaINAc-α-threonine, an α-GaINAc-L-threonine, anO-Man-L-serine, a tetra-acetyl-O-Man-L-serine, an O-GaINAc-L-serine, atri-acetyl-O-GaINAc-L-serine, a Glc-L-serine, atetraacetyl-Glc-L-serine, a fuc-L-serine, a tri-acetyl-fuc-L-serine, anO-Gal-L-serine, a tetra-acetyl-O-Gal-L-serine, a β-O-GIcNAc-L-threonine,a tri-acetyl-β-GlcNAc-L-threonine, an O-Man-L-threonine, atetra-acetyl-O-Man-L-threonine, an O-GaINAc-L-threonine, atri-acetyl-O-GaINAc-L-threonine, a Glc-L-threonine, atetraacetyl-Glc-L-threonine, a fuc-L-threonine, atri-acetyl-fuc-L-threonine, an O-Gal-L-threonine, atetra-acetyl-O-Gal-L-serine, a β-N-acetylglucosamine-O-serine,α-N-acetylgalactosamine-O-threonine, fluorescent amino acids such asthose containing naphthyl or dansyl or 7-aminocoumarin or7-hydroxycoumarin side chains, photocleavable or photoisomerizable aminoacids such as those containing azobenzene or nitrobenzyl Cys, Ser or Tyrside chains, p-carboxy-methyl-L-phenylalanine, homoglutamine,2-aminooctanoic acid, p-azidophenylalanine, p-benzoylphenylalanine,p-acetylphenylalanine, m-acetylphenylalanine, 2,4-diaminobutyric acid(DAB) and the like. The invention includes unprotected and acetylatedforms of the above. (See also, for example, WO 03/031464 A2, entitled“Remodeling and Glycoconjugation of Peptides”; and, U.S. Pat. No.6,331,418, entitled “Saccharide Compositions, Methods and Apparatus fortheir synthesis;” Tang and Tirrell, J. Am. Chem. Soc. (2001) 123:11089-11090; and Tang et al., Angew. Chem. Int. Ed., (2001) 40:8, all ofwhich are incorporated herein by reference in their entireties).

Many of the non-natural amino acids provided above are commerciallyavailable, e.g., from Sigma Aldrich (USA). Those that are notcommercially available are optionally synthesized as provided in theexamples of US 2004/138106 A1 (incorporated herein by reference) orusing standard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York), and WO02/085923, all of which are hereby incorporated by reference.

For example, meta-substituted phenylalanines are synthesized in aprocedure as outlined in WO 02/085923 (see, e.g., FIG. 14 of thepublication). Typically, NBS (N-bromosuccinimide) is added to ameta-substituted methylbenzene compound to give a meta-substitutedbenzyl bromide, which is then reacted with a malonate compound to givethe meta substituted phenylalanine. Typical substituents used for themeta position include, but are not limited to, ketones, methoxy groups,alkyls, acetyls, and the like. For example, 3-acetyl-phenylalanine ismade by reacting NBS with a solution of 3-methylacetophenone. For moredetails see the examples below. A similar synthesis is used to produce a3-methoxy phenylalanine. The R group on the meta position of the benzylbromide in that case is —OCH₃. (See, e.g., Matsoukas et al., J. Med.Chem., 1995, 38, 4660-4669, incorporated by reference in its entirety).

In some embodiments, the design of non-natural amino acids is biased byknown information about the active sites of synthetases, e.g., externalmutant tRNA synthetases used to aminoacylate an external mutant tRNA.For example, three classes of glutamine analogs are provided, includingderivatives substituted at the nitrogen of amide (1), a methyl group atthe γ-position (2), and a N-Cy-cyclic derivative (3). Based upon thex-ray crystal structure of E. coli GInRS, in which the key binding siteresidues are homologous to yeast GInRS, the analogs were designed tocomplement an array of side chain mutations of residues within a 10 Åshell of the side chain of glutamine, e.g., a mutation of the activesite Phe233 to a small hydrophobic amino acid might be complemented byincreased steric bulk at the Cy position of Gln.

For example, N-phthaloyl-L-glutamic 1,5-anhydride (compound number 4 inFIG. 23 of WO 02/085923) is optionally used to synthesize glutamineanalogs with substituents at the nitrogen of the amide. (See, e.g., King& Kidd, J. Chem. Soc., 3315-3319, 1949; Friedman & Chatterrji, J. Am.Chem. Soc. 81, 3750-3752, 1959; Craig et al., J. Org. Chem. 53,1167-1170, 1988; and Azoulay et al., Eur. J. Med. Chem. 26, 201-5, 1991,all of which are hereby incorporated by reference in their entireties).The anhydride is typically prepared from glutamic acid by firstprotection of the amine as the phthalimide followed by refluxing inacetic acid. The anhydride is then opened with a number of amines,resulting in a range of substituents at the amide. Deprotection of thephthaloyl group with hydrazine affords a free amino acid as shown inFIG. 23 of WO 2002/085923.

Substitution at the γ-position is typically accomplished via alkylationof glutamic acid. (See, e.g., Koskinen & Rapoport, J. Org. Chem. 54,1859-1866, 1989, hereby incorporated by reference). A protected aminoacid, e.g., as illustrated by compound number 5 in FIG. 24 of WO02/085923, is optionally prepared by first alkylation of the aminomoiety with 9-bromo-9-phenylfluorene (PhflBr) (see, e.g., Christie &Rapoport, J. Org. Chem. 1989, 1859-1866, 1985, hereby incorporated byreference) and then esterification of the acid moiety usingO-tert-butyl-N,N′-diisopropylisourea. Addition of KN(Si(CH₃)₃)₂regioselectively deprotonates at the α-position of the methyl ester toform the enolate, which is then optionally alkylated with a range ofalkyl iodides. Hydrolysis of the t-butyl ester and Phfl group gave thedesired γ-methyl glutamine analog (Compound number 2 in FIG. 24 of WO02/085923, hereby incorporated by reference).

An N-Cy cyclic analog, as illustrated by Compound number 3 in FIG. 25 ofWO 02/085923, is optionally prepared in 4 steps from Boc-Asp-Ot-Bu aspreviously described. (See, e.g., Barton et al., Tetrahedron Lett. 43,4297-4308, 1987, and Subasinghe et al., J. Med. Chem. 35 4602-7, 1992,each is hereby incorporated by reference). Generation of the anion ofthe N-t-Boc-pyrrolidinone, pyrrolidinone, or oxazolidone followed by theaddition of the compound 7, as shown in FIG. 25, results in a Michaeladdition product. Deprotection with TFA then results in the free aminoacids.

Trifluoroleucine (Tfl) and hexafluoroleucine (Hfl), may be synthesizedby various methods known in the art. For example,5′,5′,5′-trifluoro-DL-leucine may be synthesized in step-wise fashion byfirst diluting commercial trifluoromethyl crotonic acid with ethanol andhydrogenating it in the presence of a catalyst. Next, the mixture may berefluxed, and the ester distilled. Next,α-oximino-5′,5′,5′-trifluoroisocaproic acid may be derived by reflux anddistillation, followed by recrystalization of5′,5′,5′-trifluoro-DL-leucine. Likewise,(S)-5,5,5,5′,5′,5′-Hexafluoroleucine may be prepared fromhexafluoroacetone and ethyl bromopyruvate in multiple steps, including ahighly enantioselective reduction of the carbonyl group in an α-ketoester by bakers' yeast or by catecholborane utilizing an oxazaborolidinecatalyst. (For more details, see for example, Rennert, Anker, Biochem.1963, 2, 471; Zhang, et al., Helv. Chim. Acta 1998, 81, 174-181, R.,Prot. Sci. 7: 419-426 (1998); Hendrickson, et al., Annual Rev. Biochem.73: 147-176 (2004); U.S. Patent Application Nos. 20030108885 and20030082575, as well as copending U.S. Provisional Application No.60/571,810, all of which are hereby incorporated by reference in theirentireties). One point of novelty of the present disclosure relates toincreased thermal and chemical stability of leucine-zipper domain-richmolecules for which a fluorinated non-natural amino acid(s) has beenincorporated.

Likewise, homoproparglyglycine (HPG) and azidohomoalanine (AHA) may besynthesized by published methods. For example, according to Mangold, etal., Mutat. Res., 1989, 216, 27, which is hereby incorporated byreference in its entirety.

In addition to the above non-natural amino acids, a library of tyrosineanalogs has also been designed. Based upon the crystal structure of B.stearothermophilus TyrRS, whose active site is highly homologous to thatof the M. jannashii synthetase, residues within a 10 Å shell of thearomatic side chain of tyrosine were mutated (Y32, G34, L65, Q155, D158,A167, Y32 and D158). The library of tyrosine analogs, as shown in FIG.26 of WO 02/085923, has been designed to complement an array ofsubstitutions to these active site amino acids. These include a varietyof phenyl substitution patterns, which offer different hydrophobic andhydrogen-bonding properties. Tyrosine analogs are optionally preparedusing the general strategy illustrated by WO 02/085923 (see, e.g., FIG.27 of the publication). For example, an enolate of diethylacetamidomalonate is optionally generated using sodium ethoxide. Adesired tyrosine analog can then be prepared by adding an appropriatebenzyl bromide followed by hydrolysis.

Exemplary Molecules

Essentially any protein (or portion thereof) that includes annon-natural amino acid, e.g., an non-natural amino acid comprising amoiety where a chemical moiety is attached, such as an aldehyde- orketo-derivatized amino acid, or an non-natural amino acid that includesa chemical moiety (and any corresponding coding nucleic acid, e.g.,which includes one or more selector codons) can be produced using thecompositions and methods herein. No attempt is made to identify thehundreds of thousands of known proteins, any of which can be modified toinclude one or more non-natural amino acid, e.g., by tailoring anyavailable mutation methods to include one or more appropriate degeneratecodons in a relevant translation system. Common sequence repositoriesfor known proteins include GenBank EMBL, DDBJ and the NCBI. Otherrepositories can easily be identified by searching on the internet.

Typically, the proteins are, e.g., at least about 60%, 70%, 75%, 80%,90%, 95%, or at least about 99% or more identical to any availableprotein (e.g., a therapeutic protein, a diagnostic protein, anindustrial enzyme, or portion thereof, and the like), and they compriseone or more non-natural amino acid.

In one aspect, a composition includes at least one protein with at leastone, e.g., at least about two, three, four, five, six, seven, eight,nine, or at least about ten or more unnatural amino acids, e.g., anunnatural amino acid comprising a moiety where a saccharide moiety canbe attached, or an unnatural amino acid that includes a saccharidemoiety, and/or which include another unnatural amino acid. The unnaturalamino acids can be the same or different, e.g., there can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnaturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein substituted with the unnatural amino acid, e.g., an unnaturalamino acid comprising a moiety where a saccharide moiety can beattached, or an unnatural amino acid that includes a saccharide moiety.For a given protein with more than one unnatural amino acids, theunnatural amino acids can be identical or different (e.g., the proteincan include two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different, or a combination of multiple unnatural amino acidsof the same kind with at least one different unnatural amino acid.

A “target molecule,” “target protein,” or “target polypeptide,” and thelike as used herein generally refer to any naturally occurring orsynthetic (artificial) therapeutic, diagnostic, bio-molecule, peptides,polypeptides, or proteins that can be modified as discussed by thepresent invention. Some examples of target molecules include, but arenot limited to, e.g., α-1 antitrypsin, Angiostatin, Antihemolyticfactor, antibodies (including an antibody or a functional fragment orderivative thereof selected from: Fab, Fab′, F(ab)2, Fd, Fv, ScFv,diabody, tribody, tetrabody, dimer, trimer or minibody), angiogenicmolecules, angiostatic molecules, Apolipoprotein, Apoprotein,Asparaginase, Adenosine deaminase, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, Angiotensin family members,Bone Morphogenic Protein (BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6,BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-15, etc.); C—X—C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1α, Monocyte inflammatoryprotein-1β, RANTES, I309, R83915, R91733, HCC1, T58847, D31065, T64262),CD40 ligand, C-kit Ligand, Ciliary Neurotrophic Factor, Collagen, Colonystimulating factor (CSF), Complement factor 5a, Complement inhibitor,Complement receptor 1, cytokines, (e.g., epithelial NeutrophilActivating Peptide-78, GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-1δ, MCP-1),deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin(“EPO”, representing a preferred target for modification by theincorporation of one or more non-natural amino acid), Exfoliating toxinsA and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast GrowthFactor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF,Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog proteins(e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor(HGF), Hepatitis viruses, Hirudin, Human serum albumin, Hyalurin-CD44,Insulin, Insulin-like Growth Factor (IGF-I, IGF-II), interferons (e.g.,interferon-α, interferon-β, interferon-γ, interferon-ε, interferon-ζ,interferon-η, interferon-κ, interferon-λ, interferon-τ, interferon-

, interferon-ω), glucagon-like peptide (GLP-1), GLP-2, GLP receptors,glucagon, other agonists of the GLP-1R, natriuretic peptides (ANP, BNP,and CNP), Fuzeon and other inhibitors of HIV fusion, Hurudin and relatedanticoagulant peptides, Prokineticins and related agonists includinganalogs of black mamba snake venom, TRAIL, RANK ligand and itsantagonists, calcitonin, amylin and other glucoregulatory peptidehormones, and Fc fragments, exendins (including exendin-4), exendinreceptors interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, etc.), I-CAM-1/LFA-1, KeratinocyteGrowth Factor (KGF), Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones(e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras, Raf,Met, etc.), Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B,and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit, Signaltranscriptional activators and suppressors (p53, Tat, Fos, Myc, Jun,Myb, etc.), Soluble complement receptor I, Soluble I-CAM 1, Solubleinterleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14,15), soluble adhesion molecules, Soluble TNF receptor, Somatomedin,Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e.,Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE),Steroid hormone recetors (such as those for estrogen, progesterone,testosterone, aldosterone, LDL receptor ligand and corticosterone),Superoxide dismutase (SOD), Toll-like receptors (such as Flagellin),Toxic shock syndrome toxin (TSST-1), Thymosin α 1, Tissue plasminogenactivator, transforming growth factor (TGF-α, TGF-β), Tumor necrosisfactor β (TNF β), Tumor necrosis factor receptor (TNFR), Tumor necrosisfactor-α (TNF α), transcriptional modulators (for example, genes andtranscriptional modular proteins that regulate cell growth,differentiation and/or cell regulation), Vascular Endothelial GrowthFactor (VEGF), virus-like particle, VLA-4/VCAM-1, Urokinase, signaltransduction molecules, estrogen, progesterone, testosterone,aldosterone, LDL, corticosterone amidase, amino acid racemase, acylase,dehalogenase, dioxygenase, CD4OL/CD40, diarylpropane peroxidase,epimerase, epoxide hydrolase, esterase, isomerase, kinase, glucoseisomerase, glycosidase, glycosyl transferase, haloperoxidase,monooxygenase, lipase, lignin peroxidase, nitrile hydratase, nitrilase,protease, phosphatase, subtilisin, trnasaminase, nuclease, and manyothers.

Target molecules include transcriptional modulators, signal transductionmolecules and oncogene products, which may be found in prokaryotes,viruses, and eukaryotes, including fungi, plants, yeasts, insects, andanimals, including mammals, providing a wide range of therapeutictargets. It will be appreciated that expression and transcriptionalactivators regulate transcription by many mechanisms, e.g., by bindingto receptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA.

Some examples of transcriptional modulators or expression activatorsinclude but are not limited to: cytokines, inflammatory molecules,growth factors, their receptors, and oncogene products, e.g.,interleukins (e.g., IL-1, IL-2, IL-8, etc.), interferons, FGF, IGF-I,IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD4OL/CD40,VLA-4NCAM-1, ICAM-1/LFA-1, and hyalurin/CD44; signal transductionmolecules and corresponding oncogene products, e.g., Mos, Ras, Raf, andMet; and transcriptional activators and suppressors, e.g., p53, Tat,Fos, Myc, Jun, Myb, Rel, and steroid hormone receptors such as those forestrogen, progesterone, testosterone, aldosterone, the LDL receptorligand and corticosterone.

For modification of antibodies, the non-natural amino acid residue(s)may be placed at any location or position in the antibody structure,depending on the desired goal. For example, the non-natural amino acidresidue may be placed in the Fab variable region, the Fc region, or inanother location that interacts with the Fc region of the antibody. Inother embodiments, the non-natural amino acid residue may be placed inthe binding interface of the antibody, or the V_(H) region. In certainembodiments, the modified antibody exhibits an increase or decrease inits ability to kill one or more targets. In particular, an antibody withincreased ability to kill one or more targets, or with reduced sideeffects may be desired.

In other embodiments, the non-natural amino acid(s) confer enhancedbinding affinity to an Fc-receptor and/or to C1q of the complementsystem. In particular, a modified antibody may have an altered (e.g.,enhanced) affinity and/or specificity for an antigen or a proteinbinding partner (e.g., C1q of the complement and/or the Fc receptor onmacrophages, etc.). For example, modification of a molecule may increaseor decrease its antibody-dependent cell-mediated cytotoxicty (ADCC)function, or complement fixation activity. In other examples,modification of a particular molecule may increase or decrease itsability to bind another molecule of natural counter structure (such asan antibody).

Another class of proteins able to be modified as disclosed hereininclude enzymes (e.g., industrial enzymes) or portions thereof. Examplesof enzymes include, but are not limited to, e.g., amidases, amino acidracemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Still another class of proteins that may be modified as disclosed hereininclude vaccine proteins e.g., in proteins from infectious fungi, e.g.,Aspergillus, Candida species; bacteria, particularly E. coli, whichserves a model for pathogenic bacteria, as well as medically importantbacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g.,pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods(e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas,Giardia, etc.); viruses such as (+) RNA viruses (examples includePoxviruses e.g., vaccinia; Picornaviruses, e.g., polio; Togaviruses,e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNAviruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV;Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNAviruses (Reoviruses, for example), RNA to DNA viruses, i.e.,Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such asHepatitis B.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targetmolecules.

Some target molecules that can be modified as disclosed herein arecommercially available (see, e.g., the Sigma BioSciences catalogue andprice list), and the corresponding protein sequences and genes and,typically, many variants thereof, are well-known (see, e.g., Genbank).

Typically, the target molecules are proteins that are, e.g., at leastabout 60%, 70%, 75%, 80%, 90%, 95%, or at least about 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more non-natural amino acid.

Any of the exemplary target molecules disclosed herein or otherwise canbe modified according to methods described herein and may result inaltering one or more therapeutic, diagnositic, or enzymatic propertiesof the target protein. Examples of therapeutically relevant propertiesinclude serum half-life, shelf half-life, stability, immunogenicity,therapeutic activity, detectability (e.g., by the inclusion of reportergroups (e.g., labels or label binding sites)) in the non-natural aminoacids, specificity, reduction of LD50 or other side effects, ability toenter the body through the gastric tract (e.g., oral availability), orthe like. Examples of relevant diagnostic properties include shelfhalf-life, stability (including thermostability), diagnostic activity,detectability, specificity, or the like. Examples of relevant enzymaticproperties include shelf half-life, stability, specificity, enzymaticactivity, production capability, resistance to at least one protease,tolerance to at least one non-aqueous solvent, or the like.

Multiprotein Complexes

Another aspect of the invention provides a method for generating animmunoconjugate target molecule comprising an antibody (or functionalfragment/derivative thereof) and one or more therapeutic moieties, themethod comprising: (1) incorporating one or more non-natural aminoacid(s) at specified position(s) of the antibody using any of thesuitable subject methods; (2) contacting the antibody with the one ormore therapeutic moieties to form a conjugate that attaches the one ormore therapeutic moieties to the non-natural amino acid(s) of theantibody.

The therapeutic moieties may be the same or different, may be conjutatedto the same or different non-natural amino acids, and may be cleaveableunder one or more conditions selected from: mild or weak acidicconditions (e.g. about pH 4-6, including about pH 5), reductiveenvironment (e.g. the presence of a reducing agent), divalent cations,or optionally, heat. Additional aspects of the invention provide for animmunoconjugate target molecule produced by any of the suitable subjectmethods. Non-natural amino acids can also be used to join two or moretarget molecules or target molecule sub-units with uniquefunctionalities. For example, bispecific antibodies may be generated bylinking two target molecule antibodies (or functional parts thereof orderivatives thereof, such as Fab, Fab′, Fd, Fv, ScFv fragments, etc.)through non-natural amino acids incorporated therein.

Although the electrophilic moiety (e.g., a keto moiety, an aldehydemoiety, and/or the like) and nucleophilic moiety described herein in thecontext of attaching sugar or other chemical moieties to proteins, thesame set of electrophilic and nucleophilic moieties may be used to jointwo protein molecules, such as two antibody molecules.

Thus the instant invention provides methods for synthesis ofmulti-protein conjugates comprising target molecules. These methodsinvolve, in some embodiments, incorporating into a first target protein(e.g., a first antibody) a first non-natural amino acid that comprises afirst reactive group; and contacting the first target protein with asecond target protein (e.g., a second antibody) comprising a secondnon-natural amino acid that comprises a second reactive group, whereinthe first reactive group reacts with the second reactive group, therebyforming a covalent bond that attaches the second target protein to thefirst target protein.

The first reactive group comprises, in some embodiments, anelectrophilic moiety (e.g., a keto moiety, an aldehyde moiety, and/orthe like), and the second reactive group comprises a nucleophilicmoiety. In some embodiments, the first reactive group comprises anucleophilic moiety and the second reactive group comprises anelectrophilic moiety (e.g., a keto moiety, an aldehyde moiety, and/orthe like). For example, an electrophilic moiety is attached to thenon-natural amino acid of the first antibody, and the nucleophilicmoiety is attached to the non-natural amino acid of the second antibody.

Different functional domains of different target proteins may be linkedtogether through similar fashion to create novel proteins with novelfunctions (e.g., novel transcription factors with unique combination ofDNA binding and transcription activation domains; novel enzymes withnovel regulatory domains, etc.).

Exemplary Methods of Altering Molecules

The following means for deleting, substituting, adding or otherwiseincorporating amino acid residues may be used with non-natural aminoacid residues or naturally occurring amino acid residues, depending onthe desired outcome of each round of mutation or modification, as wellas the overall goal relating to modifying the target molecule.Non-natural amino acids may be incorporated according to specific aminoacid residue (e.g., by replacing all or nearly all positions of aparticular amino acid in the polypeptide), or site-specifically at adesired amino acid position.

With regard to amino acid residue specific incorporation, one generalapproach to modifying the target molecule comprises replacing several orall but one of a particular selected amino acid residue in the targetmolecule. In certain embodiments, the selected amino acid residue ismethionine. In at least one embodiment, every methionine amino acidresidue in a target molecule is replaced by gene mutation with anothernaturally occurring or non-natural amino acid residue. Thus, in certainembodiments, the polynucleotide is altered or modified in order tochange the nucleic acid sequence of a particular naturally occurringamino acid codon to a non-natural amino acid codon or a stop codon (orother nonsense codon) in order to allow incorporation of a non-naturalamino acid at a selected location in the target molecule. Next, theremaining amino acid residue(s) is/are replaced with a non-natural aminoacid during fermentation. Fementation allows for reduced manufacturingcosts, compared with chemical synthesis of molecules.

In certain embodiments, the non-natural amino acid corresponds to thenaturally occurring amino acid that it is replacing in the targetmolecule. In other embodiments, the non-natural amino acid codon doesnot correspond in chemical structure to the naturally occurring aminoacid codon that is being replaced in the target molecule. In certainembodiments, particularly where the non-natural amino acid doescorrespond to the naturally occurring amino acid that it is replacing inthe target molecule, the endogenous tRNA and/or aminoacyl tRNAsynthetase machinery may be used for incorporation of the non-naturalamino acid into the target molecule. In some embodiments, this methodwould rely on manufacturing in cells (such as auxotrophic host cells)that are unable or deficient in the naturally occurring amino acid thatis being replaced. Thus, during protein translation, the correspondingnon-natural amino acid is present in the culture medium (with or withoutthe corresponding naturally occurring amino acid selected to bereplaced) and the non-natural amino acid is incorporated at thenaturally occurring amino acid position that is the intended target forreplacement.

In certain other methods, non-natural amino acids may be incorporated asadditional amino acids, rather than as replacement amino acids, in thetarget molecule.

In certain embodiments where the selected amino acid residue ismethionine, azidohomoalanine or homoproparglyglycine, or othernon-natural amino acids may be substituted for the remaining methioninein the target molecule. Preferably, the target molecule retains theability to properly fold. Using this particular method ofresidue-specific incorporation, the multiple different target moleculesmay be utilized with success. Since ultimately, every specific naturallyoccurring amino acid residue in a particular amino acid family or typewill be substituted or replaced with another amino acid (whethernaturally occurring or non-natural), preferable amino acid residuefamilies to select for substitution include those in which few naturallyoccurring amino acids are present in the target molecule. For example,most preferred target molecules have few methionine or tryptophanresidues present and such amino acid types may be easily substituted orreplaced with a non-natural amino acid or other naturally occurringamino acid with a lower likelihood for disruption of the structure orfunction of the target molecule.

In one exemplary embodiment, a target molecule may have up to about 10,about 9, about 8 about 7, about 6, about 5, about 4, about 3, about 2 orabout 1 substitution(s) without disrupting the structure or function ofthe target molecule. In certain embodiments, the location of thesesubstitutions may also be considered. For instance, the substitution(s)should preferably not occupy a position in the active site for receptorbinding or other intermolecular action for the target molecule.Likewise, the substitution(s) should preferably not occupy a keystructural position unless the non-natural amino acid or naturallyoccurring replacement amino acid is chemically or structurallycompatible with those functional properties. In the event that thenon-natural or replacement naturally occurring amino acid is notcompatible, a codon of the the target molecule may be modified at thepolynucleotide level in order to encode for another amino acid (eithernaturally occurring or non-natural). Preferably, the substitution isconservative, i.e. retains the proper structure and function of thetarget molecule. Thus, methionine residues may be preferably replacedwith threonine, isoleucine, or leucine prior to replacing any remainingmethionine residues with a non-natural amino acid.

In certain embodiments in which only a single non-natural amino acid isdesired in a target molecule, then all of the methionine (or otherselected amino acid type) are substituted with other naturally occurringamino acids and one methionine amino acid residue is retained (orintroduced, if it doesn't already exist) at the desired non-naturalposition in the target molecule. Subsequently, a non-natural or otherreplacement amino acid is incorporated at the single methionine aminoacid residue position. As one of skill in the art would appreciate, thismethod may be employed for any particular amino acid type other thanmethionine.

The location of the one remaining natural amino acid residue that isreplaced by the non-natural amino acid may be any desired location forwhich the properties of the non-natural amino acid are beneficial (forexample, at the amino terminus).

In certain embodiments, in order to maintain the proper structure and/orfunction of the target molecule, the substitution of specific amino acidtypes (such as methionine) may also be accompanied by the substitutionof other amino acids that interact with the substituted amino acids,particularly for folding.

Following incorporation of the non-natural amino acid into the targetmolecule, a chemical moiety may be attached to the molecule, therebyforming a conjugate. Such methods of modifying target molecules withnon-natural amino acids enables highly specific incorporation, highlyefficient incorporation, and results in high yields if modified targetmolecules.

With regard to site-specific incorporation of non-natural amino acids,manipulation of transcriptional and/or translational machinery may berequired for increased efficiency of incorporation of a non-naturalamino acid. For example, manipulation of an aminoacyl-tRNA synthetaseand/or an aminoacyl-tRNA may be necessary in order to achievesite-specific incorporation of an non-natural amino acid. In addition,modifying the editing function of an aminoacyl tRNA synthetase may alsoprovide for increased efficiency and/or increased specificity forincorporation of a particular non-natural amino acid.

Thus, the promiscuity of some aminoacyl-tRNA synthetases (whether wildtype or mutant) may be exploited toward certain non-natural amino acidsthat bear structural resemblance to the specific natural amino acidcounterpart(s).

Furthermore, auxotrophic host cells may be utilized in order to increasethe efficiency of incorporation of the non-natural amino acid, whetherby site-specific or residue-specific incorporation. Auxotrophic hostcells are mutant cells that are unable to synthesize a particularorganic compound required for its growth and can only grow if thecompound is taken up from the growth media. When the media contains anon-natural amino acid (instead of or in addition to the naturallyoccurring amino acid counterpart), the auxotrophic host cell utilizesthe non-natural amino acid and incorporates it into the polypeptidechain. Auxotrophic host cells may be used in concert with manipulatedmachinery (such as mutant aminoacyl tRNAs and/or mutant aminoacyl tRNAsynthetases) for increased efficiency of incorporation of non-naturalamino acids.

Well over 100 non-coded amino acids (all ribosomally acceptable) havebeen reportedly introduced into proteins using other methods (see, forexample, Schultz et al., J. Am. Chem. Soc., 103: 1563-1567, 1981;Hinsberg et al., J. Am. Chem. Soc., 104: 766-773, 1982; Pollack et al.,Science, 242: 1038-1040, 1988; Nowak et al., Science, 268: 439-442,1995, all of which are hereby incorporated by reference in theirentireties) any or all of these referenced analogs may be used in thesubject methods for efficient incorporation of the analogs into proteinproducts. In general, the method of the instant invention can be used toincorporate amino acid analogs into protein products either in vitro orin vivo.

Furthermore, the target molecule can have one or more non-natural aminoacid residues at any particular position in the protein, and thenon-natural amino acid residues may be the same or different from eachother. In certain aspects, a composition of the present inventionincludes at least one protein with one or more non-natural amino acids,including at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten or more non-natural amino acid residues that may be anycombination of the seame or different from each other. Typically, thetarget molecules (e.g., proteins) may be at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% or more identical to any available target protein (e.g., atherapeutic protein, a diagnostic protein, etc.).

One of the surprising results of the present invention includes thefinding that different penultimate N-terminal (amino terminal)non-natural amino acid residues affect cellular processing of a moleculein which the N-terminal amino acid is a non-natural amino acid. Examplesof this effect are demonstrated herein. For example, in one embodiment,the non-natural amino acid codon encoding the amino acid located at theamino terminus of the polypeptide is cleaved during translationalprocessing, likely due to peptidase activity. Thus, in certainembodiments, the amino terminal non-natural amino acid codon will beretained at a greater efficiency when the second position, orpenultimate amino acid residue position, is also altered to a codon thatencodes a non-natural amino acid. These changes may be conducted in anymanner outlined herein, whether at a nucleic acid level or amino acidlevel.

The N-terminus (amino terminus) may be altered by adding a non-naturalamino acid, or by replacing the native amino acid residue (typically amethionine) with a non-natural amino acid. In particular, as describedin the Figures inter alia, specific amino acid residues at thepenultimate N-terminal position can support efficient retention orremoval of the N-terminal non-natural amino acid residue. Furthermore,unsaturated side chains found on some non-natural amino acids (such asazidohomoalanine and homoproparglyglycine) may be incorporated withlittle or no side reactions with the natural amino acids. (Kiick et al.,PNAS USA 99: 19-24 (2002); Wu, et al., Angew. Chem. Int. Ed. Eng. 43:3928-3932 (2004)).

In one exemplary embodiment, using the methods disclosed herein, amutant interferon-β conjugate was generated with azidohomoalanine (AHA)or homoproparglyglycine (HPG) incorporated at the amino terminus, aswell as the following amino acid mutations or substitutions: S2E, C17S,M36I, I40F, I44L, M62I, M117T. Thus, the target molecule interferon-βhad every methionine amino acid residue altered to another naturallyoccurring amino acid residue, with the exception of the initiatormethionine residue, which was altered to AHA. In addition, other aminoacid positions were altered to other naturally occurring amino acids.Multiple naturally occurring amino acid residues were selected foraltering the wild type sequence of the interferon-β target molecule.

In another exemplary embodiment, using the methods disclosed herein, amutant interferon-β conjugate was generated with azidohomoalanine (AHA)or homoproparglyglycine (HPG) incorporated at the amino terminus, aswell as the following amino acid mutations or substitutions: S2E, M36I,I40F, I44L, M62I, M117T. Thus, the target molecule interferon-β hadevery methionine amino acid residue altered to another naturallyoccurring amino acid residue, with the exception of the initiatormethionine residue, which was altered to AHA. In addition, other aminoacid positions were altered to other naturally occurring amino acids.I40 and I44 were altered to maintain bioactivity, since these residuesinteract with the internal methionine. The penultimate S2 residue wasaltered to eliminate methionine aminopeptidase cleave of the N-terminalAHA.

Other amino acid mutations or substitutions for the target interferon-βmolecule were conducted individually and/or combinatorially based onsequence comparisons of various species of interferon-β and/orinterferon-a molecules. Since the human interferon-β molecule containedonly 4 methionine residues in the wild type sequence (at positions 1,36, 62, and 117), and since it was desired that the chemical moiety(PEG) would be attached at the amino terminus of the molecule,methionine was selected as the amino acid to be replaced. Studyingsequences of the interferon molecules, the methionine at position 36 inhuman interferon-β was isoleucine in the corresponding dog sequence;alanine in the corresponding mouse sequence; threonine in thecorresponding rat sequence; and histidine in the human interferon-αsequence. Likewise, for the methionine located at amino acid position 62in the human interferon-β sequence, isoleucine was present in thecorresponding chicken sequence at position 62, leucine was present inthe corresponding Australian echidna sequence at position 62, leucinewas present in the corresponding human interferon-α-1 sequence (13), andvaline was present in the corresponding human interferon-α-1 sequence(6). Finally, for the methionine located at amino acid position 117 ofthe human interferon-β molecule, valine was present in the correspondingmonkey sequence at position 117, threonine and serine were present inother species at position 117, and aspartic acid, asparagines, andserine were present at position 117 in other human interferon sequences.Thus, these amino acids were first selected as the first candidates formutation and/or substitution at the corresponding methionine residues inthe human interferon-β molecule.

Additionally, once certain desired amino acid residues or amino acidpositions were identified based on the sequence comparisons, the energycalculations were conducted for various amino acid alterations to thosesites. In light of these analyses, multiple interferon-β mutationsand/or substitutions were conducted at the following amino acidpositions (amino terminus is position 1, such that M1AHA indicates thatthe methionine at position 1 is altered to AHA or azidohomoalanine, allothers follow the same format): M1AHA, M1HPG, S2H, S2E, S2Q, S2Y, S2F,S2K, M36T, M36A, M36I, M36V, M62Q, M62S, M62T, M62H, M62N, M62Y, M62F,M62I, M62A, M62L, M62G, M117any, M117S, M117T, M117Y, M117G, M62L-I40L,M62I-I40E-I44L (“Chicken triple” or “triple”), M62I-I40E-I44L-M117T,M62I-I40E-I44L-M117S, M36A-M62I-I40F-I44L, M36T-M62I-I40E-I44L,M36T-M62I-I40E-I44L-M117T, M36T-M62I-I40F-I44L-M117S, M62L-I40L,M36T-M117I (“TI,” wherein TI may comprise further mutations and/orsubstitutions), M36T-M117T (“TT,” wherein TT may comprise furthermutations and/or substitutions), TI-S2K, TI-S2Q, TI-S2Y, TI-S2F, TI-S2E,TI-S2H, TT-S2K, TT-S2Y, TT-S2F, TT-S2E, TT-S2H, TT-S2Q.

The M1AHA, S2E, C17S, M36I, I40F, I44L, M62I, M117T mutant interferon-βmolecule containing these amino acid substitutions retained the aminoterminal AHA, was easily purified and refolded properly (includingdisulfide bond formation). Additionally, the mutant interferon-βmolecule was efficiently PEGylated with poly(ethylene) glycol (10K) andpoly(ethylene) glycol (20K), and will be PEGylated with poly(ethylene)glycol (40K) using a [3+2] copper catalyzed cycloaddition between theazide moiety and the alkyne moiety. The mutant interferon-β PEGylatedconjugate was structurally stable and retained full biologicalfunctional activity both in vitro and in vivo. Details of the mutantinterferon-β conjugate are set forth in the Examples herein.

In Vitro Incorporation

In general, any means known in the art for generating transcripts can beemployed to synthesize proteins with amino acid analogs or naturallyoccurring amino acid residues. For example, any in vitro transcriptionsystem or coupled transcription/translation systems can be used togenerate a transcript of interest, which then serves as a template forprotein synthesis. Alternatively, any cell, engineered cell/cell line,or functional components (lysates, membrane fractions, etc.) that iscapable of expressing proteins from nucleic acid materials can be usedto generate a transcript. These means for generating a transcript willtypically include such components as RNA polymerase (T7, SP6, etc.) andco-factors, nucleotides (ATP, CTP, GTP, UTP), necessary transcriptionfactors, and appropriate buffer conditions, as well as at least onesuitable DNA template, but other components may also be added foroptimized reaction conditions.

In certain aspects of the present invention, target molecules may beidentified and/or modified by “DNA shuffling,” or “gene shuffling,”which may comprise point mutations, gene duplications and/or geneticrecombination. Gene shuffling may occur to some degree in nature, and isa successful laboratory procedure used in vitro or in vivo, that maymimic the natural evolutionary processes of mutation and recombinationon an accelerated scale. The technique may be used to evolve targetmolecules, including proteins and in particular enzymes or antibodies,to possess novel specificities, characteristics or activities.

For example, gene shuffling may occur by a first round of error-pronePCR, by generating an expression library or by introducing a particularnon-natural or naturally occurring amino acid residue in a host cellline, which results in random or selected mutations. The pool or libraryof mutated variants may then be submitted to random fragmentation andPCR-based reassembly to generate a population of full-length recombinedvariants. In addition to or alternatively to, the pool or library ofmutated PCR products may be expressed in a host cell that incorporates aparticular amino acid residue either randomly or selectively atparticular locations, thereby generating a round of modification for thetarget molecule of interest. Next, screening or testing the populationof variants leads to identification and isolation of particular mutantclones with improved functions or characteristics. The selected clonesmay subsequently be submitted for any number of additional rounds of“gene shuffling.” In at least certain cases, multiple rounds aresufficient to obtain optimal variants, as the particular selectedcharacteristics may be enhanced upon each successive round. In at leastsome instances, both coding and non-coding genes or gene fragments areresponsible for the enhanced characteristics or activities.

In other instances, a bacteriophage may be created for expression of alibrary containing a non-natural amino acid, where the bacteriophagegenome has been codon optimized to eliminate a particular codon thatwill be used for the incorporation of a non-natural amino acid in thebacterial host cell in which the phage library will be expressed. In atleast one embodiment, a library of mutant or variant molecules can beexpressed in a host cell line in which a codon has been introduced thatencodes a non-natural amino acid. For additional details, see forexample, Stemmer, Proc. Nat'l. Acad. Sci. USA, 91: 10747-10751 (1994),hereby incorporated by reference in its entirety.

In another exemplary embodiment, a bacteriophage is created forexpressing a library containing a non-natural amino acid, in which thebacteriophage genome has been optimized to eliminate a particular codonthat will be used for the incorporation of a non-natural amino acid inthe bacterial host cell expressing the phage.

In another exemplary embodiment, a library of a target molecule, such asScFv, such as any combinatorial library of heavy and lightimmunoglobulin chains, or such as a randomized antigen binding library(including a phage library) may be expressed in a host cell thatincorporates a non-natural amino acid at a particular codon andsubsequently introduce that codon either randomly or at particularlocations in the library of molecules. Thus, expressing the library inthe host cell would incorporate the non-natural amino acid. Next, thelibrary may be subjected to antigen binding selection to identify orisolate a particular target molecule.

In certain aspects of the invention, a target molecule may be altered ormodified for selection of a particular characteristic by chemical and/orsite-directed mutagenesis and/or multi-site incorporation. Chemicalmutagenesis may include subjecting or treating a target molecule with amutagenic agent. Mutagenic agents may function in a variety of ways,including increasing the “mispairing” ability, increasing frameshiftmutations, or damaging or altering a base. Mutagenic agents are wellknown in the art and may include base analog mutagens (such as5-bromo-deoxyuridine), alkylators (such as ethyl methane sulfonate,methyl methane sulfonate, diethylsulfate and nitrosoguanidine),chemicals causing oxidative deamination (such as nitrous acid), as wellas ultraviolet (UV) light.

Site-directed mutagenesis may involve PCR or non-PCR basedmodifications. Site-directed mutagenesis may allow for mutations of aspecific amino acid residue with a specific codon substitution, deletionor addition. In addition, a set of random mutations over a gene regionor entire gene may be accomplished by random and extensive mutagenesis(also called targeted random, region-specific, or library mutagenesis).Site-directed mutagenesis may be in vitro or in vivo.

Site-directed mutagenesis may be accomplished by a number of approaches.In particular, one approach involves using an oligonucleotidecomplementary to part of a single-stranded DNA template but containingan internal mismatch to direct the mutation. This approach may be usedfor single as well as multi-site mutations, insertions and deletions.Another approach involves replacing the region to be mutated in thetarget molecule previously obtained by ligation of a number of syntheticoligonucleotides. Following either of these approaches, the mutant ormodified target molecules may undergo sequencing to verify the desiredmutations have occurred.

Site-directed mutagenesis may be accomplished by using a singlemutagenic primer, or multiple mutagenic primers that are annealed to thesingle-stranded template, extended briefly with Klenow fragment, andused to transfect a host (such as a bacterial or yeast cell). In oneparticular method, the mutagenic primer or primers may extend around theentire plasmid containing the desired sequence to be mutated. Followingthis “all the way around” technique, the new strand may be ligated. Ifmultiple primers are used, at least one primer typically is used toprotect the mismatch mutation after extension and ligation. Anothertechnique involving a single primer is the “gapped duplex” technique,which utilizes a single-stranded region formed by annealing the templatewith a restriction fragment from the vector itself. This allows the 5′end of the oligonucleotide to be protected after extension and ligation.The template used for site-directed mutagenesis may be double-strandedor single-stranded, circular or linear, or any combination of these. Formore details for particular techniques, see for example, Carter,Biochem. J., 237:1-7 (1986); Bain, et al. J. Am. Chem. Soc. 111:8013-8014 (1989); Wang et al, Proc. Nat'l. Acad. Sci. USA 100:1 (2003);Ling and Robinson, Analy. Biochem. 254: 157-178 (1997), all of which arehereby incorporated by reference in their entireties.

In addition, point mismatch repair, or mutagenesis usingrepair-deficient host strains is further embodied by the presentinvention. Deletion mutagenesis, restriction-selection andrestriction-purification, mutagenesis by total gene synthesis,double-strand break repair, and other methods known in the art may beemployed.

As further described herein, error-prone PCR may be used to alter ormodify a target molecule, including a protein, at the genetic level. Forexample, PCR may be performed under conditions that allow for lowcopying fidelity of the DNA polymerase, and a high rate of pointmutations results in the entire PCR product. Further, recursive ensemblemutagenesis may be used in which an algorithm for protein mutagenesis isused to produce diverse populations of phenotypically related mutantswhose members differ in amino acid sequence.

In one of the embodiments, a target molecule such as an antibody and/orantibody fragment containing non-natural amino acids can be directlysynthesized chemically using solid phase synthesis and ligationtechnologies, or using in vitro translation/expression. For example theintact antibody or its fragments can also be expressed using a varietyof well-established protein expression systems including E. coli,yeasts, insect (e.g., baculo-virus system), and mammalian cells.

In another preferred embodiment, two or more analogs may be used in thesame in vitro or in vivo translation system, with or without utilizingO-tRNA/O-RS pairs. Utilizing O-tRNA/O-RS pairs may be more easilyaccomplished when a natural amino acid is encoded by four or morecodons. However, for amino acids encoded by only two codons, one can bereserved for the natural amino acid, while the other is “shared” by oneor more amino acid analog(s). These analogs may resemble only onenatural amino acid (for example, different phenylalanine analogs), orresemble different amino acids (for example, analogs of phenylalanineand tyrosine).

For in vitro use, one or more O-RSs of the instant invention can berecombinantly produced and supplied to any available in vitrotranslation systems (such as the commercially available Wheat GermLysate-based PROTEINSCRIPT-PRO™, Ambion's E. coli system for coupled invitro transcription/translation; or the rabbit reticulocyte lysate-basedRETIC LYSATE IVT™ Kit from Ambion). Optionally, the in vitro translationsystem can be selectively depleted of one or more natural AARSs (by, forexample, immunodepletion using immobilized antibodies against naturalAARS) and/or natural amino acids so that enhanced incorporation of theanalog can be achieved. Alternatively, nucleic acids encoding there-designed M-RSs may be supplied in place of recombinantly producedAARSs. The in vitro translation system may also be supplied with theanalogs to be incorporated into mature protein products.

Although in vitro protein synthesis usually cannot be carried out on thesame scale as in vivo synthesis, in vitro methods can yield hundreds ofmicrograms of purified protein containing amino acid analogs. Suchproteins have been produced in quantities sufficient for theircharacterization using circular dichroism (CD), nuclear magneticresonance (NMR) spectrometry, and X-ray crystallography. Thismethodology can also be used to investigate the role of hydrophobicity,packing, side chain entropy and hydrogen bonding in determining proteinstability and folding. It can also be used to probe catalytic mechanism,signal transduction and electron transfer in proteins. In addition, theproperties of target molecules can be modified using this methodology.For example, photocaged proteins can be generated that can be activatedby photolysis, and novel chemical handles have been introduced intotarget molecules for the site specific incorporation of optical andother spectroscopic probes.

In vivo Incorporation

The development of a general approach for the incorporation ofnon-natural amino acids into target molecules in vivo, directly from thegrowth media, would greatly enhance the power of non-natural amino acidmutagenesis. For example, the ability to synthesize large quantities ofproteins containing heavy atoms would facilitate protein structuredetermination, and the ability to site-specifically substitutefluorophores or photocleavable groups into proteins in living cellswould provide powerful tools for studying protein function in vivo.Alternatively, one might be able to enhance the properties of proteinsby providing building blocks with new functional groups, such as aketo-containing amino acid.

In certain embodiments herein, one or more AARS of the instant inventioncan be supplied to a host cell (prokaryotic or eukaryotic) as nucleicacid material, such as coding sequences on plasmids or viral vectors,which may optionally integrate into the host genome and constitutivelyor inducibly express the re-designed AARSs. A heterologous or endogenoustarget molecule can be expressed in such a host cell, at the presence ofsupplied non-natural amino acids. The protein products can then bepurified using any art-recognized protein purification techniques, ortechniques specially designed for the target molecule.

In one particular embodiment, for site-specific and/or multisiteincorporation of non-natural amino acids, a procedure described in U.S.Pat. No. 6,586,207 may be used, the entire content of which isincorporated herein by reference. Briefly, U.S. Pat. No. 6,586,207provides general methods for producing a modified target molecule,wherein the target molecule is modified by replacing a selected aminoacid with a desired non-natural amino acid. In certain embodiments, themethod relates to producing a modified polypeptide, comprising:

-   -   a. providing a host cell in a medium, the host cell comprising:    -   i. a vector having a polynucleotide sequence encoding an        aminoacyl-tRNA synthetase for an amino acid analogue; and    -   ii. a vector having a polynucleotide sequence encoding a        polypeptide molecule of interest so as to produce a host vector        system; wherein the vectors of (i) and (ii) may be the same or        different,    -   b. replacing the medium with a medium which has the desired        amino acid analogue or adding the desired amino acid analogue to        the medium, wherein the desired amino acid analogue is selected        from the group consisting of an analogue that comprises side        chain functionalities different from its corresponding natural        amino acid, an analogue that is an optical isomer of the        corresponding natural amino acid, an analogue that is a        hydrophobic amino acid analogue, and an analogue that comprises        fluorinated, electroactive, conjugated, azido, carbonyl, alkyl,        or unsaturated side chain functionalities; and any amino acid        that may be utilized efficiently by the AARS encoded on the        polynucleotide    -   c. growing the host cell in the medium which has the desired        amino acid analogue under conditions so that the host cell        expresses the polypeptide molecule of interest and the desired        amino acid analogue is incorporated into the polypeptide        molecule of interest thereby producing the modified polypeptide.

According to this method, expression of an aminoacyl-tRNA synthetaseresults in an increase in the activity of the aminoacyl-tRNA synthetase.This method is partially based on the discovery that incorporation ofnon-natural non-natural amino acids into polypeptides can be improved incells that express or overexpress aminoacyl-tRNA synthetases (AARSs)that recognize such non-natural amino acids as substrates. “Improvement”as referred to herein, includes either increasing the scope ofnon-natural amino acids (i.e., kinds of non-natural amino acids) thatcan be incorporated, or by increasing the yield of the modified targetmolecule. Expression of the aminoacyl-tRNA synthetase increases thelevel of aminoacyl-tRNA synthetase activity in the cell. The increasedactivity leads to an increased rate of incorporation of non-naturalamino acids into the growing peptide, thereby increasing the rate ofsynthesis of the target molecule, and thereby increasing the quantity ofpolypeptides containing such non-natural amino acids.

The nucleic acids encoding the aminoacyl-tRNA synthetase, and/or thenucleic acids encoding the tRNA molecule, and/or the nucleic acidsencoding the polypeptide of interest (antibody or its fragment), may belocated in the same or different vectors. The vectors may includeexpression control elements which direct the production of the AARS, thetRNA, and the target molecule. The expression control elements (i.e.,regulatory sequences) can include inducible promoters, constitutivepromoters, secretion signals, enhancers, transcription terminators, andother transcriptional regulatory elements.

For both in vivo as well as in vitro incorporation of non-natural aminoacids into a target molecule, any combination of multisite and/orsite-specific incorporation (including addition or substitution) may beutilized in making a modified target molecule. In one particular method,multiple amino acid residues or positions of a particular amino acidfamily are selected and replaced with alternative naturally occurringamino acids, which preferably allow for retention of function of thetarget molecule. Next, some or all of these selected amino acid residuesare replaced with one or more non-natural amino acid(s). In anotherparticular method, a naturally occurring amino acid residue may be addedto a particular protein such that it is the sole amino acid residue ofthat particular family, or only one of a few in the target molecule.Subsequently, the added amino acid residue is replaced with one or morenon-natural amino acid residues. In certain embodiments, the non-naturalamino acid corresponds to or is in the same amino acid family as thenaturally occurring amino acid it replaced.

Host Cells and Translation Systems

Certain embodiments disclosed herein can be practiced within a cell,which enables production levels of target molecules to be made forpractical purposes. In preferred embodiments, the cells used areculturable cells (i.e., cells that can be grown under laboratoryconditions). Suitable cells include mammalian cells (human or non-humanmammals), bacterial cells, and insect cells, etc.

One example includes PFENEX™ technology, which is a cell line usingPseudomonas fluorescens-based cell lines that increase cellularexpression while maintaining certain solubility and activitycharacteristics due to its use of different pathways in the metabolismof certain sugars compared to E.coli.

In addition, other auxotrophic host cell lines include K10 based Pheauxotrophic strain (AF), DH10B based Phe auxotrophic strain (AF),Phe/Trp double auxotrophic strains (AFW), Phe/Trp/Lys triple auxotrophicstrains (AFWK), and a Met auxotroph (M15MA on M15 background).

Cells that may be used to practice certain embodiments disclosed hereininclude auxotrophic host cells (whether prokaryotic or eukaryotic).Auxotrophic cells may exhibit single, double, triple, quadruple, orgreater levels of auxotrophy (each level of auxotrophy indicates aparticular organic compound that the organism is unable to synthesize orotherwise lacks and must be supplied to the cell). Certain embodimentsdisclosed herein expressly do not utilize an auxotrophic host cell.Insofar as an auxotrophic host cell is not used, another cell or cellcomponents may be used to practice certain embodiments disclosed herein.Other embodiments may use one, two, three, or more different auxotrophichost cells that may be from the same or different strains or organisms.

Host cells may be genetically engineered (e.g., transformed, transducedor transfected) with the vectors of this disclosure, which can be, forexample, a cloning vector or an expression vector. The vector can be,for example, in the form of a plasmid, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (From et al., PNAS. USA 82, 5824 (1985)),infection by viral vectors, high velocity ballistic penetration by smallparticles with the nucleic acid either within the matrix of small beadsor particles, or on the surface (Klein et al., Nature 327, 70-73(1987)). Berger, Sambrook, and Ausubel provide a variety of appropriatetransformation methods.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms.

Some examples of host cells that may be useful include but are notlimited to (e.g., mammalian cells, yeast cells, bacterial cells, plantcells, fungal cells, archaebacterial cells, insect cells, and/or thelike). Some examples of specific host cells include E.coli, Pseudomonas,S. cerivisiae, etc.

In certain embodiments, the non-natural amino acid is provided byintroducing additional nucleic acid construct(s) into the translationsystem, wherein the additional nucleic acid construct(s) encode one ormore proteins required for biosynthesis of the non-natural amino acid.

In one embodiment, the translation system is a cell, and the methodfurther comprises disabling one or more genes encoding any endogenoustRNA that forms Watson-Crick base-pairing with the codon(s) at thespecified position(s). In one embodiment, the translation system is acell, and the method further comprises inhibiting one or more endogenousAARS that charges tRNAs that form Watson-Crick base-pairing with thecodon(s) at the specified position(s).

Also provided by the invention are compositions that include atranslation system. The translation systems may include one or both ofan external mutant or modified tRNA (M-tRNA) and/or an external mutantor modified aminoacyl tRNA synthetase (M-RS). In embodiments thatutilize M-tRNA and/or M-RS, may be derived from a species different fromthat of the cell.

In certain embodiments, the translation system comprises more than twodifferent subject polynucleotides or nucleic acid constructs. Each ofthe polynucleotides, or nucleic acid constructs is capable of carrying adifferent non-natural amino acid. In certain embodiments, the firstpolynucleotide further comprises a first promoter sequence controllingthe expression of the M-tRNA. In certain embodiments, the secondpolynucleotide further comprises a second promoter sequence controllingthe expression of the modified AARS. The M-RS may have a relaxedsubstrate specificity, or the M-RS may be capable of charging the M-tRNAwith an non-natural amino acid.

In certain embodiments, the M-tRNA is from a yeast, and the cell is anE. coli bacterium. In certain embodiments, the M-RS and the M-tRNA arefrom the same organism, and the organism is different from that of thecell. In certain embodiments, the M-RS and the M-tRNA are from a yeast,and the cell is an E. coli bacterium.

In certain embodiments, the expression and/or function of an endogenoustRNA homologous to the tRNA is impaired or abolished. In certainembodiments, the expression of the endogenous tRNA is impaired/abolishedby inhibiting the function of the endogenous tRNA's cognate AARS,thereby impairing/abolishing the charging of the endogenous tRNA. Incertain embodiments, the expression of the endogenous tRNA is abolishedby deleting the gene encoding the endogenous tRNA.

Under certain circumstances, the modified tRNA interacts with the wobbledegenerate codon with an affinity at 37° C. of at least about 1.0kcal/mole, or 1.5 kcal/mole, or even 2.0 kcal/mole more favorably thanthe interaction between its unmodified version and the wobble degeneratecodon.

In enzyme kinetics, k_(cat) is a first-order rate constant correspondingto the slowest step or steps in the overall catalytic pathway. Thek_(cat) represents the maximum number of target molecules of substratewhich can be converted into product per enzyme target molecule per unittime (which occurs if the enzyme is “saturated” with substrate), andthus is often referred to as the turnover number. The K_(m) is anapparent dissociation constant and is related to the enzyme's affinityfor the substrate; it is the product of all the dissociation andequilibrium constants prior to the first irreversible step in thepathway. Often, it is a close measure of the enzyme-substratedissociation constant. The k_(cat)/K_(m) is a second-order rate constantwhich refers to the free enzyme (not enzyme-substrate complex) and isalso a measure of the overall efficiency of the enzyme catalysis and isalso referred to as the specificity constant.

In certain embodiments, the external mutant synthetase has improved orenhanced enzymatic properties, e.g., the K_(m) is higher or lower, thek_(cat) is higher or lower, the value of k_(cat)/K_(m) is higher orlower or the like, for the non-natural amino acid compared to anaturally occurring amino acid, e.g., one of the 20 known amino acids.The Km of the M-RS is preferably equal to or lower for the non-naturalamino acid than for the corresponding wild type natural amino acid.

In certain embodiments, the k_(cat)/K_(m) values of the M-RS, orexogenous AARS, may range from 3-fold, 5-fold, 10-fold, 25-fold,50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold,385-fold, 400-fold higher than for the naturally occurring amino acid.

In some exemplary embodiments, typical Km values for different aminoacids with M-RS may range from approximately 15 microM, 20 microM, 30microM, 50 microM, 75 microM, 100 microM, 150 microM, 200 microM, 300microM, 400 microM, 440 microM, 500 microM, 1000 microM, 1500 microM,2000 microM, 3000 microM, 4000 microM, 5000 microM, 6000 microM, orgreater or any value therebetween.

Likewise, the k_(cat) values of the M-RS or exogenous AARS, ispreferably equal to or higher for the non-natural amino acid than forthe natural amino acid. For example, k_(cat) values for different aminoacids with the corresponding M-RS may range from approximately 0.002sec⁻¹, 0.0018 sec⁻¹, 0.0015 sec⁻¹, 0.014 sec⁻, 0.1 sec⁻, 0.3 sec⁻¹, 1sec⁻¹, 3 sec⁻¹, 5 sec⁻¹, 8 sec⁻¹, 10 sec⁻¹, 13.3 sec⁻, 15 sec⁻¹, orhigher.

Thus, the k_(cat)/Km of the M-RS or exogenous AARS, is optimally equalto or higher for the non-natural amino acid than for the natural wildtype amino acid. Typical k_(cat)/Km values may range from approximately0.0001 M⁻¹ s⁻¹, 0.0003 M⁻¹ s⁻¹, 0.005 M⁻¹ s⁻¹, 0.05 M⁻¹ s⁻¹, 0.5 M⁻¹s⁻¹, 0.547 M⁻¹ s⁻¹, 1 M⁻¹ s⁻¹, 5 M⁻¹ s⁻¹, 10 M⁻¹ s⁻¹, 20 M⁻¹ s⁻¹, 30 M⁻¹s⁻¹, 32 M⁻¹ s⁻¹, 500 M⁻¹ s⁻¹, 600 M⁻¹ s⁻¹, 1000 M⁻¹ s⁻¹, 5000 M⁻¹ s⁻¹,11000 M⁻¹ s⁻¹.

In certain embodiments, the rate of the ATP-PPi exchange reactioncatalyzed by AARSs in the presence of amino acids can be measured forthe molecules of the present invention. It is generally considered thatthe aminoacyl-tRNA is formed through a two step process. In the firststep, the amino acid is accepted by the synthetase and is adenylated,which results in a release of pyrophosphate (PPi). In the second step,the proper tRNA is accepted by the synthetase, and the amino acidresidue is transferred to the 2′ or 3′ OH of the 3′-terminal residue ofthe tRNA. Thus, measurement of the ATP-PPi exchange rate will indicatethe formation of the aminoacyl-tRNA for a particular amino acid, aparticular tRNA, or a particular AARS, depending on the desired goal.

In certain embodiments, the M-tRNA interacts with the wobble degeneratecodon with an affinity at 37° C. of at least about 1.0 kcal/mole, 1.5kcal/mole, 2.0 kcal/mole, 2.5 kcal/mole, 3.0 kcal/mole, 3.5 kcal/mole,4.0 kcal/mole, 4.5 kcal/mole, 5.0 kcal/mole or greater (or any valuetherebetween) favorably than the interaction between its unmodifiedversion and the wobble degenerate codon.

The methods of the invention can be practiced within a cell, whichenables production levels of proteins to be made for practical purposes.Because of the high degree of conservation of the genetic code and thesurrounding molecular machinery, method of the invention can be used inmost cells. In at least one embodiment, the cells used are culturablecells (i.e., cells that can be grown under laboratory conditions).

The present invention includes host cells and cell lines alreadygenerated (including auxotropic prokaryotic strains and/or eukaryoticstrains). In one embodiment, the host cell is generally capable ofincorporating a non-natural amino acid into a peptide or polypeptidechain. In at least one embodiment, the host cell is capable ofselectively or preferentially incorporating a non-natural amino acidinto a peptide or polypeptide chain. In at least one embodiment, thehost cell is capable of exclusively incorporating a non-natural aminoacid into a peptide or polypeptide chain.

In the host-vector system, the production of an aminoacyl-tRNAsynthetase can be controlled by a vector which comprises expressioncontrol elements that direct the production of the aminoacyl-tRNAsynthetase. Preferably, the production of aminoacyl-tRNA synthetase isin an amount that enables efficient incorporation of the specifiednon-natural amino acid into the target molecule.

In the host-vector system, the production of an aminoacyl-tRNAsynthetase can be controlled by a vector which comprises expressioncontrol elements that direct the production of the aminoacyl-tRNAsynthetase. Preferably, the production of aminoacyl-tRNA synthetase isin an amount in excess of the level of naturally occurringaminoacyl-tRNA synthetase, such that the activity of the aminoacyl-tRNAsynthetase is greater than naturally occurring levels.

In the host-vector system, the production of an antibody, fragment, orother target molecule can be controlled by a vector that comprisesexpression control elements for producing the modified target molecule.In certain aspects, the target molecule so produced is in an amount inexcess of the level produced by a naturally occurring gene encoding thetarget molecule.

The host-vector system can constitutively express the AARS and induce toexpress the target molecule (e.g., antibody) by contacting thehost-vector system with an inducer, such asisopropyl-β-D-thiogalactopyranoside (IPTG). The host-vector system canalso be induced to express the aminoacyl-tRNA synthetase and/or theprotein of interest by contacting the host-vector system with aninducer, such as IPTG. Other inducers include stimulation by an externalstimulation such as heat shock.

In one embodiment, the host-vector system is grown in media lacking thenatural amino acid and supplemented with a non-natural non-natural aminoacid. It is in this media that the target polypeptide is induced forexpression, thereby producing a modified target molecule that hasincorporated at least one non-natural amino acid. This method issuperior to existing methods as it improves the efficiency ofincorporating non-natural amino acids into target molecules, and itincreases the quantity of the modified target molecules so produced.

In another embodiment, the host-vector system may be used to regulate orinduce the expression of a target molecule in host cells where suchinduction is desirable. In particular, the target molecule may be undercontrol of an inducible promoter, or alternatively, under the control ofa strong promoter when the polynucleotide contains one or more stopcodon, frameshift codon, or bias codon at a specific position thatprevents the target molecule from being efficiently translated.

The translational machinery of the host cell will read through thespecified codon, effectively inducing expression of the target molecule,in the presence of the host-vector system and upon addition of thenon-natural amino acid. This type of inducible expression may increasethe ability to manufacture high levels of toxic proteins, and may beparticularly useful in mammalian cells wherein inducible proteinsynthesis is limited. Thus, protein products, such as monoclonalantibodies, are expressed constitutively. In this manner, an induciblesystem of protein synthesis enables increased expression of moleculesthat would otherwise be toxic to the host cells. Moreover, itfacilitates incorporation of non-natural amino acids in mammalian cellswhen the non-natural amino acid itself is toxic.

Other methods for modifying target molecules include constructingexpression libraries (e.g., U.S. Pat. Nos. 5,783,431; 5,824,485, herebyincorporated by reference in their entireties). Libraries may becomposed of cDNA or genomic sequences from a single organism or species,or multiple organisms or species. The sequences are operably linked toproper regulatory sequences in an expression cassette. The sequences mayalso be generally optionally randomly concatenated to further enhancediversity. Expression libraries may be preselected or prescreened for aparticular sequence that encodes a functional product. Libraries mayalso be generated that are biased towards particular sequences thatencode target molecules with particularly desired activities.

Another method of incorporating one or more non-natural amino acidresidues is by utilizing bias codons for which there is a low abundanceof corresponding tRNA such that the presence of a bias codonsignificantly slows translation of the protein. The bias codon specifiesthe non-natural amino acid through the introduction of a tRNA thatdecodes the bias codon in the host cell. The tRNA is subsequentlyaminoacylated by an aminoacyl-tRNA synthetase specific for thenon-natural amino acid.

In one embodiment, the codon that specifies a non-natural amino acid isa codon that is decoded by a two box set of tRNAs, a four box set oftRNAs, or a six box set of tRNAs. This includes, but is not limited to,serine, arginine, and leucine. The specified codon may be selected fromone box that will not base pair by Watson and Crick or Wobble with tRNAsfor the same amino acids. For example, serine tRNAs that decode UCU,UCC, UCA, and UCG codons, will not base pair with the serine AGU or AGCcodons. Thus, the non natural amino acid, used by a modified SerRS, maybe specified by the AGU (Wobble) codon. All other serine residues in theprotein of interest would be specified by UCU, UCC, UCA, and UCG. Inthis way, the non natural amino acid would be specifically incorporatedat the AGU codon.

In one embodiment, the tRNA may be one that is normally used by adifferent amino acyl tRNA synthetase, but whose aminoacylation beenchanged due to modification or mutation of the tRNA at a criticalidentity determining position. For example, the Gln tRNA, with certainmodification including a change to the opal anticodon, is aminoacylatedby the TrpRS. Conversely, the Trp tRNA may be used by the GInRS todecode an Amber stop codon.

In one embodiment, the AARS is a chimeric fusion of 2 differentsynthetases such that the aminoacylation function of one synthetase isfused to the tRNA binding and identity elements of another. This willresult in the aminoacylation of a tRNA with an incorrect amino acid, andthe incorporation of that amino acid at the codon normally reserved foranother amino acid. The chimeric AARS may be further modified toincorporate a non natural amino acid. The derivation of the chimericAARS may utilize computational biology, gene shuffling, or other domainshuffling strategies.

In the case of using an amber or wobble stop codon, such codon may beplaced anywhere in the target molecule, depending on the desired goal.For example, such codon may be placed at the preferred site forattaching a chemical moiety, such as polyethylene glycol. Followinginsertion of the stop codon, a non-natural amino acid residue (such asp-bromo-phenylalanine) is incorporated at the codon site by any processdescribed herein or known in the art. For instance, the non-naturalamino acid may be incorporated via an auxotrophic host cell, by M-RS, byM-tRNA molecules, or any combination thereof. If utilizing anauxotrophic host cell, the host cell may be a single auxotroph (i.e.deficient in or unable to synthesize a single particular amino acid,therefore able to incorporate the single corresponding non-natural aminoacid from the culture media) or a multiple auxotroph (i.e. incapable ofsynthesizing more than one amino acid, thereby capable of incorporatingmore than one non-natural amino acid from the culture media). Thus, thenon-natural amino acid is specifically incorporated without disruptingother residues, and without the need to screen large numbers of mutants.

As one of skill in the art would appreciate that any of theaforementioned methods to modify or alter a target molecule mayincorporate radioactive, doped or other tags or markers in the processof modification.

Generation of AARS by Mutagenesis and Selection/Screening

In certain embodiments, the AARS capable of charging a particular M-tRNAwith a particular non-natural amino acid can be obtained by mutagenesisof the AARS to generate a library of candidates, followed by screeningand/or selection of the candidate AARS's capable of their desiredfunction. Such M-RS and M-tRNA molecules may be used for in vitro or invivo production of desired target molecule with modified non-naturalamino acids.

Libraries of M-RSs can be generated using various mutagenesis techniquesknown in the art. For example, the M-RSs can be generated bysite-specific mutations, random mutations, diversity generatingrecombination mutations, chimeric constructs, and by other methodsdescribed herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, e.g., thataminoacylate a mutant tRNA (M-tRNA) in the presence of an non-naturalamino acid and a natural amino acid, includes: introducing a positiveselection or screening marker, e.g., an antibiotic resistance gene, orthe like, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one codon, whose translation (optionally conditionally) dependson the ability of a candidate M-RS to charge the M-tRNA (with either anatural and/or a non-natural amino acid); growing the plurality of cellsin the presence of a selection agent; identifying cells that survive (orshow a specific response) in the presence of the selection and/orscreening agent by successfully translating the codon in the positiveselection or screening marker, thereby providing a subset of positivelyselected cells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried. Preferably, the cells do not contain any functional endogenoustRNA/RS pair that can help to translate the codon. The endogenoustRNA/RS pair may be disabled by gene deletion and/or RS inhibitors.

Since many essential genes of the cell likely also contain codons thatrely on the ability of the M-RS to charge the M-tRNA at the absence offunctional endogenous translational machinery, in certain embodiments noextra positive selection markers are needed for the positive selectionprocess—the survival of the cell can be used as a confirmation ofpositive selection.

In other embodiments, positive selection markers may be used; such as achloramphenicol acetyltransferase (CAT) gene. Optionally, the positiveselection marker is a β-lactamase gene. In another aspect the positivescreening marker comprises a fluorescent or luminescent screening markeror an affinity based screening marker (e.g., a cell surface marker).

In a similar embodiment, a cell-free in vitro system may be used to testthe ability of M-RS to charge M-tRNA in a positive screening. In oneembodiment, negatively selecting or screening the pool for active RSs(optionally mutants) that preferentially aminoacylate the M-tRNA in theabsence of the non-natural amino acid includes: introducing a negativeselection or screening marker with the pool of active (optionallymutant) RSs from the positive selection or screening into a plurality oftranslational systems, wherein the negative selection or screeningmarker comprises at least one codon (e.g., codon for a toxic markergene, e.g., a ribonuclease barnase gene), whose translation depends onthe ability of a candidate M-RS to charge the M-tRNA; and identifyingthe translation system that shows a specific screening response in afirst media supplemented with the non-natural amino acid and a screeningor selection agent, but fails to show the specific response in a secondmedia supplemented with the natural amino acid and the selection orscreening agent, thereby providing surviving cells or screened cellswith the at least one recombinant M-RS.

In one aspect, the concentration of the selection (and/or screening)agent is varied. In some aspects the first and second organisms aredifferent. Thus, the first and/or second organism optionally comprises:a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, ayeast, an archaebacterium, a eubacterium, a plant, an insect, a protist,etc. In other embodiments, the screening marker comprises a fluorescentor luminescent screening marker or an affinity based screening marker.

In a related aspect, methods for producing a recombinant mutant tRNA(M-tRNA) include: (a) generating a library of mutant tRNAs derived fromat least one tRNA, from a first organism; (b) selecting (e.g.,negatively selecting) or screening the library for (optionally mutant)tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from asecond organism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced mutant RS (M-RS), thereby providing atleast one recombinant M-tRNA; wherein the at least one recombinantM-tRNA recognizes a degenerate codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe M-RS.

Methods for generating specific M-tRNA/M-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introduced mutantRS (M-RS), thereby providing at least one recombinant M-tRNA. The atleast one recombinant M-tRNA preferentially recognizes a degeneratecodon and is not efficiently recognized by the RS from the secondorganism and is preferentially aminoacylated by the M-RS. The methodalso includes (d) generating a library of (optionally mutant) RSsderived from at least one aminoacyl-tRNA synthetase (RS) from a thirdorganism; (e) selecting or screening the library of mutant RSs formembers that preferentially aminoacylate the at least one recombinantM-tRNA in the presence of an non-natural amino acid and a natural aminoacid, thereby providing a pool of active (optionally mutant) RSs; and,(f) negatively selecting or screening the pool for active (optionallymutant) RSs that preferentially aminoacylate the at least onerecombinant M-tRNA in the absence of the non-natural amino acid, therebyproviding the at least one specific M-tRNA/M-RS pair, wherein the atleast one specific M-tRNA/M-RS pair comprises at least one recombinantM-RS that is specific for the non-natural amino acid and the at leastone recombinant M-tRNA. Specific M-tRNA/M-RS pairs produced by themethods are included. Additionally, such methods include wherein thefirst and third organism are the same (e.g., Methanococcus jannaschii).

The various methods of the invention (above) optionally comprise whereinselecting or screening comprises one or more positive or negativeselection or screening, e.g., a change in amino acid permeability, achange in translation efficiency, and a change in translationalfidelity. Additionally, the one or more change is optionally based upona mutation in one or more gene in an organism in which an externalmutant tRNA-tRNA synthetase pair are used to produce such protein.Selecting and/or screening herein optionally comprises wherein at least2 codons within one or more selection gene or within one or morescreening gene are used. Such multiple codons are optionally within thesame gene or within different screening/selection genes. Additionally,the optional multiple codons are optionally different codons or comprisethe same type of codons.

Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetase (used interchangeably herein with AARS or“synthetase”) used in the methods of the invention can be a naturallyoccurring synthetase derived from a different organism, a mutated ormodified synthetase or a wholly de novo designed synthetase.

The synthetase used can recognize the desired non-natural amino acidselectively over other amino acids available to the cell. For example,when the non-natural amino acid to be used is structurally related to anaturally occurring amino acid in the cell, the synthetase should chargethe M-tRNA target molecule with the desired non-natural amino acid withan efficiency at least substantially equivalent to that of, and morepreferably at least about twice, 3 times, 4 times, 5 times or more thanthat of the naturally occurring amino acid. However, in cases in which awell-defined protein product is not necessary, the synthetase can haverelaxed specificity for charging amino acids. In such an embodiment, amixture of external mutant tRNAs could be produced, with various aminoacids or analogs.

Preferably, the modified AARS specifically or preferentially charges thenon-natural amino acid to the modified tRNA over any natural amino acid.In a preferred embodiment, the specificity constant for activation ofthe analog by the modified AARS (defined as k_(cat)/K_(m)) is equal toor greater than at least about 2-fold larger than that for the naturalamino acid, preferably about 3-fold, 4-fold, 5-fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold or more than that for the natural amino acid.

In certain embodiments, the synthetase can be designed usingcomputational techniques such as those described in Datta et al., J. Am.Chem. Soc. 124: 5652-5653, 2002, and in copending U.S. patentapplication Ser. No. 10/375,298 (or US patent application publicationUS20040053390A1, all of which are hereby incorporated by reference intheir entireties).

Domain Shuffling Design of an AARS

For an M-RS or exogenous AARS that is utilized for incorporation of anon-natural amino acid by way of a borrowed codon, the M-RS or exogenousAARS may be designed rationally by identifying the amino acid bindingdomains and tRNA identity determining domains of the first and secondAARS. In the preferred embodiment, the first and second AARS are ofrelated or homologous structure. The domains responsible may be definedand redistributed to create M-RS molecules that contain the amino acidbinding domains of one AARS and the tRNA identity elements of the other.

The shuffling of domains of the two AARS molecules of the borrowed codonmay be accomplished by using directed gene shuffling in which severalrelated AARS molecules of at least two different specificities aresubjected to PCR mediated recombination in order to generate a library.The library may subsequently be screened by methods known in the art inorder to select the M-RS or exogenous AARS of the preferred specificity.In certain embodiments, the M-RS may be generated from within the sameamino acid family, from across different amino acid families, and/orfrom different source organisms.

Computational Design of a Molecule

Specifically, in one embodiment, the subject method partly depends onthe design and engineering of a wild type molecule to a modified form.One particular method is described in more detail in US patentapplication publication US20040053390A1, the entire contents of whichare incorporated herein by reference.

Briefly, the methods described therein relate to computational tools formodifying a particular target molecule through mutation or modification.

According to the method, a rotamer library for the non-natural aminoacid is built by varying its torsional angles to create rotamers thatmight be incorporated into the target molecule of interest. Thegeometric orientation of the backbone of the non-natural amino acid isspecified by the crystallographic orientation of the backbone of thenatural substrate in the crystal structure.

The protocol may also employ a computational method to enhance theinteractions between the ligand or receptor binding site of the targetmolecule of interest. Enhancing these interactions may occur by scalingup the pair-wise energies in the energy calculations between the ligandor receptor and the amino acids allowed at the design positions on thetarget molecule. In an optimization calculation where theprotein-ligand/receptor interactions are scaled up compared to theintra-protein interactions, sequence selection is biased towardselecting amino acids to be those that have favorable interaction withthe ligand/receptor.

Available Sequence and Structural Information for Non-natural AminoAcids

In the method of the present invention, an accurate description of thetarget molecule is important for the computational design approach,since the energy calculations depend on the crystal structure for theprotein backbone descriptions. However, in many cases it may beperfectly acceptable to use a known crystal structure of a homologousprotein (for example, a homolog from a related species) or even aconserved domain to substitute for an unknown crystal structure of thetarget molecule to be modified and/or the non-natural amino acid to beincorporated. It may be preferred that the modified target moleculebinds to its corresponding ligand/receptor in the same orientation asthe unmodified target molecule, since this orientation may be importantfor the structure and/or function of the target molecule and/or itsligand/receptor.

The target molecule to be modified may be from any organism, includingprokaryotes and eukaryotes, such as bacteria, fungi, extremeophiles suchas the archebacteria, worms, insects, fish, amphibian, birds, animals(particularly mammals and particularly human) and plants.

The crystal structures of the target molecule to be modified may bederived anew or provided by known structure databases, such as theBrookhaven Protein Data Bank (PDB, see Bernstein et al., J. Mol. Biol.112: 535-542, 1977). A structure database or Molecular Modeling DataBase(MMDB) contains experimental data from crystallographic and NMRstructure determinations. The data for MMDB are obtained from theProtein Data Bank (PDB). The NCBI (National Center for BiotechnologyInformation) has cross-linked structural data to bibliographicinformation, to the sequence databases and to the NCBI taxonomy. Cn3D,the NCBI 3D structure viewer, can be used for easy interactivevisualization of molecular structures from Entrez.

The Entrez 3D Domains database contains protein domains from the NCBIConserved Domain Database (CDD). Computational biologists defineconserved domains based on recurring sequence patterns or motifs. CDDcurrently contains domains derived from two popular collections, Smartand Pfam, plus contributions from colleagues at NCBI, such as COG. Thesource databases also provide descriptions and links to citations. Sinceconserved domains correspond to compact structural units, CDs containlinks to 3D-structure via Cn3D whenever possible.

To identify conserved domains in a protein sequence, the CD-Searchservice employs the reverse position-specific BLAST algorithm. The querysequence is compared to a position-specific score matrix prepared fromthe underlying conserved domain alignment. Hits may be displayed as apairwise alignment of the query sequence with a representative domainsequence, or as a multiple alignment. CD-Search now is run by default inparallel with protein BLAST searches. While the user waits for the BLASTqueue to further process the request, the domain architecture of thequery may already be studied. In addition, CDART, the Conserved DomainArchitecture Retrieval Tool allows user to search for proteins withsimilar domain architectures. CDART uses precomputed CD-search resultsto quickly identify proteins with a set of domains similar to that ofthe query. (For more details, see Marchler-Bauer et al., Nucleic AcidsRes. 31: 383-387, 2003; and Marchler-Bauer et al., Nucleic Acids Res.30: 281-283, 2002, both of which are hereby incorporated by reference intheir entireties).

Alternatively, in certain embodiments, the exact crystal structure of aparticular target molecule is not known but its protein sequence issimilar or homologous to a known sequence with a known crystalstructure. In such instances, it is expected that the conformation ofthe target molecule will be similar to the known crystal structure ofthe homologous sequence. The known structure may, therefore, be used asthe structure for the target molecule, or may be used to predict thestructure of the target molecule (i.e., in “homology modeling” or“molecular modeling”). As a particular example, the Molecular ModelingDatabase (MMDB) described above (see, Wang et al., Nucl. Acids Res.2000, 28:243-245; Marchler-Bauer et al., Nucl. Acids Res. 1999, 27:240-243, which are hereby incorporated by reference in their entireties)provides search engines that may be used to identify proteins and/ornucleic acids that are similar or homologous to a protein sequence(referred to as “neighboring” sequences in the MMDB), includingneighboring sequences whose three-dimensional structures are known. Thedatabase further provides links to the known structures along withalignment and visualization tools, such as Cn3D (developed by NCBI),RasMol, etc., whereby the homologous and parent sequences may becompared and a structure may be obtained for the parent sequence basedon such sequence alignments and known structures.

The homologous sequence with known 3D-structure may be at least about60%, or at least about 70%, or at least about 80%, or at least about90%, or at least about 95% identical to the target molecule of interest.

In the few cases where the structure for a particular target molecule'sgene or protein sequence may not be known or available, it is typicallypossible to determine the structure using routine experimentaltechniques (for example, X-ray crystallography and Nuclear MagneticResonance (NMR) spectroscopy) and without undue experimentation. (See,e.g., NMR of Macromolecules: A Practical Approach, G. C. K. Roberts,Ed., Oxford University Press Inc., New York (1993); Ishima and Torchia,Nat. Struct. Biol. 7: 740-743, 2000; Gardner and Kay, Annu. Rev. Bioph.Biom. 27: 357-406, 1998; Kay, Biochem. Cell. Biol. 75: 1-15, 1997; Dayieet al., Annu. Rev. Phys. Chem. 47: 243-282, 1996; Wuthrich, ActaCyrstallogr. D 51: 249-270, 1995; Kahn et al., J. Synchrotron Radiat. 7:131-138, 2000; Oakley and Wilce, Clin. Exp. Pharmacol. P. 27: 145-151,2000; Fourme et al., J. Synchrotron Radiat. 6: 834-844, 1999, all ofwhich are hereby incorporated by reference in their entireties).

Alternatively, in other embodiments, the three-dimensional structure ofa target molecule's nucleic acid or amino acid sequence may becalculated from the sequence itself and using ab initio molecularmodeling techniques already known in the art. (See e.g., Smith et al.,J. Comput. Biol. 4: 217-225, 1997; Eisenhaber et al., Proteins 24:169-179, 1996; Bohm, Biophys Chem. 59: 1-32, 1996; Fetrow and Bryant,BioTechnol. 11: 479-484, 1993; Swindells and Thorton, Curr. Opin.Biotech. 2: 512-519, 1991; Levitt et al., Annu. Rev. Biochem. 66:549-579, 1997; Eisenhaber et al., Crit. Rev. Biochem. Mol. 30: 1-94,1995; Xia et al., J. Mol. Biol. 300: 171-185, 2000; Jones, Curr. Opin.Struc. Biol. 10: 371-379, 2000 all of which are hereby incorporated byreference in their entireties). Three-dimensional structures obtainedfrom ab initio modeling are typically less reliable than structuresobtained using empirical (e.g., NMR spectroscopy or X-raycrystallography) or semi-empirical (e.g., homology modeling) techniques.However, such structures will generally be of sufficient quality,although less preferred, for use in the methods of this invention.

Methods for Predicting 3D Structure based on Sequence Homology

For target molecules to be modified that have not been crystallized orbeen the focus of other structural determinations, a computer-generatedmolecular model of the target molecule and its ligand/receptor bindingsite can nevertheless be generated using any of a number of techniquesavailable in the art.

Computer programs for performing energy minimization routines arecommonly used to generate molecular models. For example, both the CHARMM(Brooks et al. (1983) J Comput Chem 4:187-217) and AMBER (Weiner et al(1981) J. Comput. Chem. 106: 765) algorithms handle all of the molecularsystem setup, force field calculation, and analysis (see also,Eisenfield et al. (1991) Am J Physiol 261:C376-386; Lybrand (1991) JPharm Belg 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbamet al. (1990) Proteins 7:99-111; Pedersen (1985) Environ Health Perspect61:185-190; and Kini et al. (1991) J Biomol Struct Dyn 9:475-488). Inaddition, Hier Dock or Monte Carlo calculations may be employed (Datta,et al., Protein Science, 13:2693-2705 (2004). All of these citedreferences are hereby incorporated by reference in their entireties.

At the heart of these programs is a set of subroutines that, given theposition of every atom in the model, calculate the total potentialenergy of the system and the force on each atom. These programs mayutilize a starting set of atomic coordinates, the parameters for thevarious terms of the potential energy function, and a description of themolecular topology (the covalent structure). Common features of suchmolecular modeling methods include: provisions for handling hydrogenbonds and other constraint forces; the use of periodic boundaryconditions; and provisions for occasionally adjusting positions,velocities, or other parameters in order to maintain or changetemperature, pressure, volume, forces of constraint, or other externallycontrolled conditions.

Most conventional energy minimization methods use the input coordinatedata and the fact that the potential energy function is an explicit,differentiable function of Cartesian coordinates, to calculate thepotential energy and its gradient (which gives the force on each atom)for any set of atomic positions. This information can be used togenerate a new set of coordinates in an effort to reduce the totalpotential energy and, by repeating this process over and over, tooptimize the molecular structure under a given set of externalconditions.

In general, energy minimization methods can be carried out for a giventemperature, Ti, which may be different than the docking simulationtemperature, To. Upon energy minimization of the target molecule at Ti,coordinates and velocities of all the atoms in the system are computed.Additionally, the normal modes of the system are calculated. It will beappreciated by those skilled in the art that each normal mode is acollective, periodic motion with all parts of the system moving in phasewith each other and that the motion of the target molecule is thesuperposition of all normal modes. For a given temperature, the meansquare amplitude of motion in a particular mode is inverselyproportional to the effective force constant for that mode, so that themotion of the target molecule will often be dominated by the lowfrequency vibrations.

After the molecular model has been energy minimized at Ti, the system is“heated” or “cooled” to the simulation temperature, To, by carrying outan equilibration run where the velocities of the atoms are scaled in astep-wise manner until the desired temperature, To, is reached. Thesystem is further equilibrated for a specified period of time untilcertain properties of the system, such as average kinetic energy, remainconstant. The coordinates and velocities of each atom are then obtainedfrom the equilibrated system.

Further energy minimization routines can also be carried out. Forexample, a second class of methods involves calculating approximatesolutions to the constrained EOM for the protein. These methods use aniterative approach to solve for the Lagrange multipliers and, typically,only need a few iterations if the corrections required are small. Themost popular method of this type, SHAKE (Ryckaert et al. (1977) J.Comput. Phys. 23:327; and Van Gunsteren et al. (1977) Mol. Phys.34:1311, both of which are hereby incorporated by reference in theirentireties) is easy to implement and scales as O(N) as the number ofconstraints increases. An alternative method, RATTLE (Anderson (1983) J.Comput. Phys. 52:24, hereby incorporated by reference) is based on thevelocity version of the Verlet algorithm.

In other embodiments, rather than holding the identity of thenon-natural amino acid constant and varying the molecule's structure (bymodeling several different mutant structures), the subject method iscarried out using the molecular model(s) for a single modified targetmolecule (e.g., in which one more non-anchor amino acid residues arechanged) and sampling a variety of different non-natural amino acids orpotential fragments thereof, to identify analogs which are likely tosupport the molecule's structure and/or function. This approach can makeuse of coordinate libraries for non-natural amino acids (includingrotamer variants) or libraries of functional groups and spacers that canbe joined to form the side-chain of an non-natural amino acid.

There are a variety of computational methods that can be readily adaptedfor identifying the structure of non-natural amino acids that would haveappropriate steric and electronic properties to incorporate in thetarget molecule to be modified. (See, for example, Cohen et al. (1990)J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol 161: 269-288;DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989)(Spec. Publ., Roy. Soc. Chem.) 78: 182-196; Goodford et al. (1985) J.Med. Cam. 28: 849-857; DesJarlais et al. J. Med. Cam. 29: 2149-2153 allof which are hereby incorporated by reference in their entireties).Directed methods generally fall into two categories: (1) design byanalogy in which 3-D structures of known molecules (such as from acrystallographic database) are docked to the modified target moleculestructure and scored for goodness-of-fit; and (2) de novo design, inwhich the non-natural amino acid model is constructed piece-wise in themodified target molecule.

In an illustrative embodiment, the design of potential non-natural aminoacids that may function with a particular modified target moleculebegins from the general perspective of shape complimentary for thetarget molecule's structure, and a search algorithm is employed which iscapable of scanning a database of small target molecules of knownthree-dimensional structure for candidates which fit geometrically intothe substrate binding site. Such libraries can be general small targetmolecule libraries, or can be libraries directed to non-natural aminoacids or small target molecules that can be used to create non-naturalamino acids. It is not expected that the target molecules found in theshape search will necessarily be leads themselves, since no evaluationof chemical interaction necessarily be made during the initial search.Rather, it is anticipated that such candidates might act as theframework for further design, providing molecular skeletons to whichappropriate atomic replacements can be made. Of course, the chemicalcomplimentary of these target molecules can be evaluated, but it isexpected that atom types will be changed to maximize the electrostatic,hydrogen bonding, and hydrophobic interactions with the ligand-receptorbinding site. Most algorithms of this type provide a method for findinga wide assortment of chemical structures that may be complementary tothe shape of the target molecule's ligand/receptor binding site.

For instance, each of a set of small target molecules from a particulardatabase, such as the Cambridge Crystallographic Data Bank (CCDB) (Allenet al. (1973) J. Chem. Doc. 13: 119), is individually docked to themodified target molecule in a number of geometrically permissibleorientations with use of a docking algorithm. In a preferred embodiment,a set of computer algorithms called DOCK, can be used to characterizethe shape of invaginations and grooves that form the binding site. See,for example, Kuntz et al. (1982) J. Mol. Biol 161: 269-288.

The orientations are evaluated for goodness-of-fit and the best are keptfor further examination using molecular mechanics programs, such asAMBER or CHARMM. Such algorithms may provide several advantages. First,such algorithms can retrieve a remarkable diversity of moleculararchitectures. Second, the best structures have, in previousapplications to other proteins, demonstrated impressive shapecomplementarity over an extended surface area. Third, the overallapproach appears to be quite robust with respect to small uncertaintiesin positioning of the candidate atoms.

In certain embodiments, the subject method can utilize an algorithmdescribed by Goodford (1985, J. Med. Chem. 28:849-857) and Boobbyer etal. (1989, J. Med. Chem. 32:1083-1094), both of which are herebyincorporated by reference. Those papers describe a computer program(GRID) which seeks to determine regions of high affinity for differentchemical groups (termed probes) on a molecular surface. GRID provides atool for suggesting modifications to known ligands that might enhancebinding. It may be anticipated that some of the sites discerned by GRIDas regions of high affinity correspond to “pharmacophoric patterns”determined inferentially from a series of known ligands. As used herein,a pharmacophoric pattern is a geometric arrangement of features of theanticipated non-natural amino acid that is believed to be important forbinding. Goodsell and Olson (1990, Proteins: Struct Funct Genet8:195-202) have used the Metropolis (simulated annealing) algorithm todock a single known ligand into a target protein, and their approach canbe adapted for identifying suitable non-natural amino acids for dockingwith the target molecule. This algorithm can allow torsional flexibilityin the amino acid side-chain and use GRID interaction energy maps asrapid lookup tables for computing approximate interaction energies.

Yet a further embodiment of the present invention utilizes a computeralgorithm such as CLIX which searches such databases as CCDB for smalltarget molecules which can be oriented in the ligand/receptor bindingsite of the target molecule in a way that is both sterically acceptableand has a high likelihood of achieving favorable chemical interactionsbetween the candidate target molecule and the surrounding amino acidresidues. The method is based on characterizing the substrate bindingsite in terms of an ensemble of favorable binding positions fordifferent chemical groups and then searching for orientations of thecandidate target molecules that cause maximum spatial coincidence ofindividual candidate chemical groups with members of the ensemble. Thecurrent availability of computer power dictates that a computer-basedsearch for novel ligands follows a breadth-first strategy. Abreadth-first strategy aims to reduce progressively the size of thepotential candidate search space by the application of increasinglystringent criteria, as opposed to a depth-first strategy wherein amaximally detailed analysis of one candidate is performed beforeproceeding to the next. CLIX conforms to this strategy in that itsanalysis of binding is rudimentary and seeks to satisfy the necessaryconditions of steric fit by having individual groups in “correct” placesfor bonding, without imposing the sufficient condition that favorablebonding interactions actually occur. A ranked “shortlist” of targetmolecules, in their favored orientations, is produced which can then beexamined on a target molecule-by-target molecule basis, using computergraphics and more sophisticated molecular modeling techniques. CLIX isalso capable of suggesting changes to the substituent chemical groups ofthe candidate target molecules that might enhance binding. Again, thestarting library can be of non-natural amino acids or of targetmolecules which can be used to generate the side-chain of an non-naturalamino acid. The algorithmic details of CLIX is described in Lawerence etal. (1992) Proteins 12:31-41, hereby incorporated by reference in itsentirety.

Yet another embodiment of a computer-assisted molecular design methodfor identifying non-natural amino acids that may be utilized by apredetermined modified target molecule comprises the de novo synthesisof potential inhibitors by algorithmic connection of small molecularfragments that will exhibit the desired structural and electrostaticcomplementarity with the ligand/receptor binding site of the targetmolecule.

In yet another embodiment, potential non-natural amino acids can bedetermined using a method based on an energy minimization-quenchedmolecular dynamics algorithm for determining energetically favorablepositions of functional groups in the target molecule to be modified.The method can aid in the design of target molecules that incorporatesuch functional groups by modification of known amino acid andnon-natural amino acids or through de novo synthesis.

For example, the multiple copy simultaneous search method (MOSS)described by Miranker et al. (1991) Proteins 11: 29-34, hereinincorporated by reference, can be adapted for use in the subject method.To determine and characterize a local minima of a functional group inthe force field of the protein, multiple copies of selected functionalgroups are first distributed in an amino acid position of interest onthe target molecule to be modified. Energy minimization of these copiesby molecular mechanics or quenched dynamics yields the distinct localminima. The neighborhood of these minima can then be explored by a gridsearch or by constrained minimization. In one embodiment, the MOSSmethod uses the classical time dependent Hartee (TDH) approximation tosimultaneously minimize or quench many identical groups in the forcefield of the protein.

Implementation of the MOSS algorithm requires a choice of functionalgroups and a molecular mechanics model for each of them. Groups must besimple enough to be easily characterized and manipulated (3-6 atoms, fewor no dihedral degrees of freedom), yet complex enough to approximatethe steric and electrostatic interactions that the functional groupwould have in the selected position in the target molecule to bemodified. A preferred set is, for example, one in which most organictarget molecules can be described as a collection of such groups(Patai's Guide to the Chemistry of Functional Groups, ed. S. Patai (NewYork: John Wiley, and Sons, (1989), hereby incorporated by reference).This includes fragments such as acetonitrile, methanol, acetate, methylammonium, dimethyl ether, methane, and acetaldehyde.

Determination of the local energy minima in the binding site requiresthat many starting positions be sampled. This can be achieved bydistributing, for example, 1,000-5,000 groups at random inside a spherecentered on the binding site; only the space not occupied by the proteinneeds to be considered. If the interaction energy of a particular groupat a certain location with the protein is more positive than a givencut-off (e.g., 5.0 kcal/mole) the group is discarded from that site.Given the set of starting positions, all the fragments are minimizedsimultaneously by use of the TDH approximation (Elber et al. (1990) J.Am. Chem. Soc. 112: 9161-9175), hereby incorporated by reference. Inthis method, the forces on each fragment consist of its internal forcesand those due to the protein. The essential element of this method isthat the interactions between the fragments are omitted and the forceson the protein are normalized to those due to a single fragment. In thisway simultaneous minimization or dynamics of any number of functionalgroups in the field of a single protein can be performed.

Minimization is performed successively on subsets of, e.g., 100, of therandomly placed groups. After a certain number of step intervals, suchas 1,000 intervals, the results can be examined to eliminate groupsconverging to the same minimum. This process is repeated untilminimization is complete (e.g., RMS gradient of 0.01 kcal/mole/Å). Thusthe resulting energy minimized set of target molecules comprises whatamounts to a set of disconnected fragments in three dimensionsrepresenting potential side-chains for non-natural amino acids.

The next step then is to connect the pieces with spacers assembled fromsmall chemical entities (atoms, chains, or ring moieties) to formnon-natural amino acids, e.g., each of the disconnected can be linked inspace to generate a single target molecule using such computer programsas, for example, NEWLEAD (Tschinke et al. (1993) J. Med. Chem. 36: 3863,3870), herein incorporated by reference. The procedure adopted byNEWLEAD executes the following sequence of commands (1) connect twoisolated moieties, (2) retain the intermediate solutions for furtherprocessing, (3) repeat the above steps for each of the intermediatesolutions until no disconnected units are found, and (4) output thefinal solutions, each of which is a single molecule. Such a program canuse for example, three types of spacers: library spacers, single-atomspacers, and fuse-ring spacers. The library spacers are optimizedstructures of small molecules such as ethylene, benzene and methylamide.The output produced by programs such as NEWLEAD consist of a set ofmolecules containing the original fragments now connected by spacers.The atoms belonging to the input fragments maintain their originalorientations in space. The molecules are chemically plausible because ofthe simple makeup of the spacers and functional groups, andenergetically acceptable because of the rejection of solutions withvan-der Waals radii violations.

In addition, the order in which the steps of the present method areperformed is purely illustrative in nature. In fact, the steps can beperformed in any order or in parallel, unless otherwise indicated by thepresent disclosure.

Furthermore, the method of the present invention may be performed ineither hardware, software, or any combination thereof, as those termsare currently known in the art. In particular, the present method may becarried out by software, firmware, or microcode operating on a computeror computers of any type. Additionally, software embodying the presentinvention may comprise computer instructions in any form (e.g., sourcecode, object code, interpreted code, etc.) stored in anycomputer-readable medium (e.g., ROM, RAM, magnetic media, punched tapeor card, compact disc (CD) in any form, DVD, etc.). Furthermore, suchsoftware may also be in the form of a computer data signal embodied in acarrier wave, such as that found within the well-known Web pagestransferred among devices connected to the Internet. Accordingly, thepresent invention is not limited to any particular platform, unlessspecifically stated otherwise in the present disclosure.

Exemplary computer hardware means suitable for carrying out theinvention can be a Silicon Graphics Power Challenge server with 10R10000 processors, for example, running in parallel. Suitable softwaredevelopment environment includes, for example, CERIUS2 byBiosym/Molecular Simulations (San Diego, Calif.), or other equivalents.

The computational method described above has been effectively used inmodifying enzymes of the protein synthesis machinery (e.g., AARS) toallow incorporation of unnatural amino acids. The same suite ofcomputational tools can also be leveraged to design the final products(e.g., monoclonal antibodies or other therapeutics) in which theunnatural amino acids would be incorporated so as to enhance or modifytheir structural or functional properties.

Testing for Incorporation

It is further recognized that if one desired goal for modifying themolecule is to retain at least one native function, then testing of itsfunction may be conducted following each round of amino acidmodification (including substitution of one or more particular aminoacid residues). Methods of identifying incorporation of non-naturalamino acids and/or chemical moieties are well known in the art. Forexample, flow cytommetry, Northern blots, Western blots, PCR, RNAmicrosequencing, reporter assays, FLAG epitopes, binding to othermolecules (such as streptavidin), radio-label detection, colorimetricassays, RNAse protection assays, mass spectrometry (including MALDI andMALDI-TOF), chromatography (such as HPLC), NMR, IR, ELISA, fluorescentmicroscopy and any combination of these or other techniques known in theart may be implemented.

In addition to incorporating one or more members of a particular aminoacid family into the modified target molecule, it is recognized thatother amino acid residues may be physically or chemically altered(including substituted) in order to promote or retain proper molecularstructure (i.e. folding) and/or at least one native function. Forexample, it may be necessary to alter certain specific amino acidresidues that interact with residues already substituted or selected forsubstitution in the target molecule. As another example, it may benecessary to alter certain specific amino acid residues that interactwith the selected target molecule's corresponding binding partner (e.g.,receptor-ligand binding site) in addition to the other amino acidresidues chosen for substitution. Therefore, multiple amino acidresidues from multiple amino acid families may be substituted (tonaturally occurring or non-natural amino acid residues) in the selectedtarget molecule, depending on the goal of modification, as well as thenative structure of the selected target molecule.

In one of the embodiments, a protein such as an antibody and/or antibodyfragment containing non-natural amino acids can be directly synthesizedchemically using solid phase synthesis and ligation technologies, orusing in vitro translation/expression. For example the intact antibodyor its fragments can also be expressed using a variety ofwell-established protein expression systems including E. coli, yeasts,insect (e.g., baculo-virus system), and mammalian cells.

In certain embodiments, the method of site specific incorporation of nonnatural amino acids includes inducing the translation of a protein suchthat the mature, functional protein product is not expressed unless thenon natural amino acid is added to the growth medium of the cell. Insome embodiments, “mature, functional protein product” includes a geneproduct that is translated from a start codon to a stop codon. In someembodiments, “mature, functional protein product” includes a proteinproduct that is modified post-translationally, such as by glycosylation,phosphorylation, or other modification. In some embodiments, “mature,functional protein product” includes a protein that is folded in aconfiguration that allows for at least one function, including byinteraction with other target molecules (including engaging with one ormore receptors, playing a role in one or more enzymatic activities, orpairing with one or more ligands). In some embodiments, a “maturefunctional protein product” may include a precursor protein product suchas, for example, a member of the angiotensin peptide family, or theinsulin peptide family.

Nucleic Acid Constructs

In certain embodiments, the target molecule (or portion or fragmentthereof) in the methods and/or compositions of the invention is encodedby a nucleic acid. Typically, the nucleic acid comprises at least onedegenerate codon, at least about two, three, four, five, six, seven,eight, nine, or at least about ten or more degenerate codons.

In one embodiment, at least one of the modified nucleic acidconstruct(s) is operably linked to and subject to the control of apromoter, preferably an inducible promoter. In one embodiment, multiplepolynucleotides are encoded by a plasmid or plasmids. In one embodiment,a first polynucleotide further comprises a first promoter sequencecontrolling the expression of the modified tRNA. In one embodiment, thefirst promoter is an inducible promoter. In one embodiment, a secondpolynucleotide further comprises a second promoter sequence controllingthe expression of the modified AARS. In certain embodiments, the firstand second polynucleotides are present on the same target molecule.

As described herein, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences. However,one of skill in the art will appreciate that the invention is notlimited to those sequences disclosed herein. One of skill willappreciate that the present invention also provides many related andunrelated sequences with the functions described herein.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences wherein the variants hybridize toat least one disclosed sequence are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques are alsoincluded in the invention.

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular non-natural amino acid may not exist in nature, e.g., in E.coli, the invention provides such methods. For example, biosyntheticpathways for non-natural amino acids are optionally generated in E. coliby adding new enzymes or modifying existing E. coli pathways. Additionalnew enzymes are optionally naturally occurring enzymes or artificiallyevolved enzymes. For example, the biosynthesis of p-aminophenylalanine(as presented, e.g., in WO 02/085923, hereby incorporated by reference)relies on the addition of a combination of known enzymes from otherorganisms. The genes for these enzymes can be introduced into a cell,e.g., an E. coli cell, by transforming the cell with a plasmidcomprising the genes. The genes, when expressed in the cell, provide anenzymatic pathway to synthesize the desired compound. Examples of thetypes of enzymes that are optionally added are provided in the examplesbelow. Additional enzyme sequences are found, e.g., in Genbank.Artificially evolved enzymes are also optionally added into a cell inthe same manner. In this manner, the cellular machinery and resources ofa cell are manipulated to produce non-natural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, e.g., as developed by Maxygen, Inc.,is optionally used to develop novel enzymes and pathways. (See, e.g.,Stemmer 1994, Nature 370(4): 389-391; and Stemmer, 1994, Proc. Natl.Acad. Sci. USA. 91: 10747-10751, which are hereby incorporated byreference in their entireties). Similarly DesignPath™, developed byGenencor is optionally used for metabolic pathway engineering, e.g., toengineer a pathway to create a non-natural amino acid in E coli. Thistechnology reconstructs existing pathways in host organisms using acombination of new genes, e.g., identified through functional genomics,and molecular evolution and design. Diversa Corporation also providestechnology for rapidly screening libraries of genes and gene pathways,e.g., to create new pathways. One of the biosynthetic pathways mayinclude the editing function of protein translation, such that theefficiency of an AARS disclosed herein is increased by a mutant editingfunction.

Typically the non-natural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, e.g., a natural cellularamount, but not to such a degree as to affect the concentration of theother amino acids or exhaust cellular resources. Typical concentrationsproduced in vivo in this manner are about 10 mM to about 0.05 mM. Once abacterium is transformed with a plasmid comprising the genes used toproduce enzymes desired for a specific pathway and a twenty-first aminoacid, e.g., pAF, dopa, O-methyl-L-tyrosine, or the like, is generated,in vivo selections are optionally used to further optimize theproduction of the non-natural amino acid for both ribosomal proteinsynthesis and cell growth.

In some embodiments, the incorporation rates of a non-natural amino acidwere approximately 65% or greater, 70% or greater, 75% or greater, 80%or greater, 85% or greater, 90% or greater, 91% or greater, 92% orgreater, 93% or greater, 94% or greater, 95% or greater, 96% or greater,97% or greater, 98% or greater, or 99% or greater utilizing a modifiedRS.

Adding Chemical Moieties to Molecules

The addition of one or more chemical moieties to a target molecule,including a protein, can modulate protein folding, secretion, biologicalactivity, serum half-life, localization, and other properties. Theincorporation of a non-natural amino acid, e.g., a non-natural aminoacid comprising a moiety at which place a chemical moiety can beattached, or a non-natural amino acid that includes an attached chemicalmoiety, can be done to, e.g., tailor changes in protein structure and/orfunction, e.g., to change size, acidity, nucleophilicity, hydrogenbonding, hydrophobicity, accessibility of protease target sites, targetaccess to a protein moiety, etc. Proteins that include a non-naturalamino acid, e.g., a non-natural amino acid comprising a moiety where achemical moiety can be attached, or a non-natural amino acid thatincludes a chemical moiety, can have enhanced, or even entirely new,catalytic or physical properties.

For example, the following properties are optionally modified byinclusion of a non-natural amino acid joined to a chemical moiety:toxicity, biodistribution, structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalytic ability,half-life (e.g., serum half-life), ability to react with othermolecules, e.g., covalently or noncovalently, protein stability, proteinactivity, protein conformation, protein substrate specificity,protein-target binding affinity, antigen-binding ability,thermostability, protein resistance to at least one protease, proteintolerance to at least one non-aqueous environment, glycosylationpattern, phosphorylation pattern, disulfide bonding, protease cleavagesite location, metal binding ability, co-factor binding ability,cross-linking ability, solubility, cysteinylation, deamidation,acetylation, biotinylation, oxidation, glutathionylation, suiphanation,half-life in serum, immunogenicity, tissue penetration, fluorescencepegylation, multimerization ability, toxicity, biodistribution, facilityof purification, processing structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalyticactivity, ability to function as a vaccine, retard excretion fromsubject's or patient's body, redox potential, ability to react withother molecules either covalently or noncovalently, patient tolerance tosaid protein, increased efficacy of said protein in a patient, improveddelivery of said protein or protein product in a patient, increasedresistenace to peptidase, and any combination thereof.

Besides clearance through kidneys and the liver, a significantproportion of biotherapeutics are cleared through receptor-mediateddegradation. Cytokines and growth factors, when bound to theirreceptors, are internalized into cellular compartments called endosomeswhere the receptor-ligand complexes are degraded. However, those ligandsthat dissociate rapidly from their receptors in the endosome arerecycled back to the cell surface and avoid depletion, thereby elicitingincreased half-life.

Several chemical moieties, including poly(ethylene)glycol, react withfunctional groups present in the twenty naturally occurring amino acids,such as, for example, the epsilon amino group in lysine amino acidresidues, the thiol present in cysteine amino acid residues, or othernucleophilic amino acid side chains. When multiple naturally occurringamino acids react in the protein, these non-specific chemical reactionsresult in a final protein product that contains many isomers of proteinsconjugated to one or more poly(ethylene)glycol strands at differentlocations within the protein.

One advantage of certain embodiments of the present invention includesthe ability to add one or more chemical moiety (such as poly(ethylene)glycol) by incorporating non-natural amino acids that possess uniquefunctional groups that react with an activated poly(ethylene)glycolstrand by way of chemistry that is unreactive with the naturallyoccurring amino acids present in the target molecule. For example, azideand alkyne groups are unreactive with all naturally occurring functionalgroups in a protein. Thus, the non-natural amino acid may beincorporated in one or more specific sites in a target molecule wherepoly(ethylene)glycol or other modification is desired without theundesirable non-specific reactions. In certain embodiments, theparticular chemistry involved in the reaction results in a stable,covalent link between the poly(ethylene)glycol strand and the targetmolecule. In addition, such reactions may be performed in mild aqueousconditions that are not damaging to most target molecules. Thus, unlikereactions with standard polypeptides that contain highly reactivenaturally occurring amino acid residues, the reactions disclosed hereinthat utilize non-natural amino acid residues can be performed in vivo oron unpurified preparations of the target molecule due to the lack ofundesirable non-specific reactions with the biological functionalgroups.

Chemical moieties attached to natural amino acids are limited in numberand scope. By contrast, chemical moieties attached to non-natural aminoacids can utilize a significantly greater spectrum of useful chemistriesby which to attach the chemical moiety to the target molecule.

Essentially any target molecule, including any protein (or portionthereof) that includes a non-natural amino acid, e.g., a non-naturalamino acid containing a reactive site or side chain where a chemicalmoiety may attach, such as an aldehyde- or keto-derivatized amino acid,can serve as a substrate for attaching a chemical moiety. Some examplesof specific proteins are described herein inter alia, and no attempt ismade to identify every known protein which can be modified to includeone or more non-natural amino acid, e.g., by tailoring any availablemutation methods to include one or more appropriate degenerate codons ina relevant translation system. Common sequence repositories for knownproteins include GenBank EMBL, DDBJ and the NCBI.

A target molecule with an added chemical moiety is herein referred to asa “conjugate.” “Chemical moiety,” as referred to herein, may include anybiological or chemical addition or modification, or any combinationthereof, to an amino acid residue of the target molecule. Chemicalmoieties may be conjugated directly or indirectly (by way of a linker)to a non-natural amino acid or a naturally occurring amino acid in thetarget molecule.

Some examples of chemical moieties that are included in the presentinvention include but are not limited to, cytotoxins, pharmaceuticaldrugs, dyes or fluorescent labels (e.g., green-fluorescent protein orred-fluorescent protein), a nucleophilic or electrophilic group, aketone or aldehyde, azide or alkyne compounds, photocaged groups (e.g.,nitrobenzyl ethers and esters), tags (e.g., biotin), a peptide,polypeptide or protein, a glycosylation group (such as anoligosaccharide), poly(ethylene) glycol (PEG) with any molecular weight(e.g., PEG2000, PEG3350, PEG3500, PEG8000) and in any geometry (linear,branched, star, dendrimer, etc.), other poly(alkylene) glycols,poly(propylene) glycol, polyoxyethylated glycerol, polyoxyethylatedsorbitol, polyoxyethylated glucose, poly(vinyl) alcohol, metals or metalcomplexes, polyamines, imidizoles, carbohydrates (including dextran orchitosan), peptides, polypeptides, proteins, lipids, biopolymers,particles, solid supports (e.g., resin), any polymer that alters thepharmacodynamics of a target molecule, a targeting agent, an affinitygroup (such as biotin or streptavidin), any agent to which acomplementary reactive chemical group can be attached, biophysical orbiochemical probes (such as isotpically labeled amino acids, spin-labelamino acids and fluorophores, aryl iodides and bromides and anycombination of these or others. For further examples see Magliery, Med.Chem. Rev. 2005, 2, 303-323, hereby incorporated by reference in itsentirety.

The moiety may be strongly electrophilic or nucleophilic and thereby beavailable for reacting directly with the therapeutic target molecule orthe antibody or fragment thereof. Alternatively, the moiety may be aweaker electrophile or nucleophile and therefore require activationprior to the conjugation with the therapeutic molecule or the antibodyor fragment thereof. This alternative would be desirable where it isnecessary to delay activation of the chemically reactive moiety until anagent is added to the target molecule in order to prevent the reactionof the agent with the moiety. In either scenario, the moiety ischemically reactive, the scenarios differ (in the reacting with antibodyscenario) by whether following addition of an agent, the moiety isreacted directly with an antibody or fragment thereof or is reactedfirst with one or more chemicals to render the moiety capable ofreacting with an antibody or fragment thereof. In certain embodiments,the chemically reactive moiety includes an amino group, a sulfhydrylgroup, a hydroxyl group, a carbonyl-containing group, or an alkylleaving group.

Polyalkylene glycols that are particularly suitable for use in preparingthe conjugates of the invention include, but are not limited to,poly(ethylene glycols), and copolymers of ethylene oxide and propyleneoxide; particularly preferred are PEGs, and more particularly preferredare monofunctionally activated hydroxyPEGs (e.g., hydroxyPEGs activatedat a single terminus, including reactive esters ofhydroxyPEG-monocarboxylic acids, hydroxyPEG-monoaldehydes,hydroxyPEG-monoamines, hydroxyPEG-monohydrazides,hydroxyPEG-monocarbazat-es, hydroxyPEG-monoiodoacetamides,hydroxyPEG-monomaleimides, hydroxyPEG-monoorthopyridyl disulfides,hydroxyPEG-monooximes, hydroxyPEG-monophenyl carbonates,hydroxyPEG-monophenyl glyoxals, hydroxyPEG-monothiazolidine-2-thiones,hydroxyPEG-monothioesters, hydroxyPEG-monothiols,hydroxyPEG-monotriazines and hydroxyPEG-monovinylsulfones).

In certain embodiments, it may be necessary or desirable to minimize theformation of intramolecular and intermolecular crosslinking by polymers,such as PEG, during the reaction in which the polymer is attached orcoupled to the modified target molecule to form the conjugates of theinvention. Minimizing cross-linking, including intramolecular crosslinks with individual protein molecules, “dumbbell” structures, in whichone strand of polymer connects two protein molecules, and largeraggregates or gels. Minimizing these and other crosslinking reactionsmay be accomplished by using polymers that are activated at only one end(monofunctionally activated, as described above) or polymer preparationsin which the percentage of bifunctionally active (referred to as“bis-activated PEG diols” in the case of linear PEGs) ormulti-functionally activated polymers is less than about 50%, 40%, 35%,25%, 15%, 10%, 5%, or 2% (w/w). In certain embodiments, the overallPEGylation rate (that is, for at least one strand of PEG attached to thetarget molecule) is approximately 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or greater.

Particularly preferred polymers for use in preparing the conjugates ofthe present invention, which have reduced antigenicity, substantiallyreduced antigenicity, or no detectable antigenicity, aremonofunctionally activated PEGs that do not contain methoxyl groups,other alkoxyl groups or aryloxyl groups. The substitution of suchmonofunctionally activated PEGs in place of monofunctionally activatedmPEG in the synthesis of conjugates of the invention confers on theresulting conjugates an unexpectedly decreased antigenicity, i.e., adecreased ability to interact with antibodies developed against mPEGconjugates of the same bioactive component. The resultant conjugatesalso have decreased immunogenicity, i.e., decreased ability to evoke animmune response.

In certain such embodiments, the polyalkylene glycol has a molecularweight of from about 1,000 Daltons to about 100 kDa, preferably about 2kDa to about 60 kDa; about 2 kDa to about 30 kDa, about 5 kDa to about20 kDa; about 10 kDa to about 40 kDa; about 10 kDa to about 20 kDa; twobranches each with a molecular weight of about 2 kDa to about 30 kDa;and more preferably two branches, each of about 18 kDa to about 22 kDa.In one particular embodiment, the polyalkylene glycol is poly(ethylene)glycol and has a molecular weight of about 10 kDa; about 20 kDa, about30 kDa, or about 40 kDa. In specific embodiments, the PEG is a PEG 10kDa (linear or branched), a PEG 20 kDa (linear or branched), a PEG 30kDa (linear or branched), or a PEG 40 kDa (linear or branched).

Conjugates according to this aspect of the invention may comprise one ormore strands of polyalkylene glycol, in certain embodiments preferablyfrom about one to about 10 strands, from about one to about fivestrands, more preferably from about one to about three strands, and mostpreferably from about one to about two strands; in other embodimentspreferably from about five to about 100 strands, from about 10 to about50 strands and more preferably from about six to about 20 strands persubunit of high molecular weight enzyme proteins. In a particularlypreferred such embodiment, the polyalkylene glycol used in the conjugatecomprises one or two strands of a monofunctionally activatedpoly(ethylene glycol) (e.g., a reactive ester of a hydroxyPEG-monoacid,a hydroxyPEG-monoaldehyde, a hydroxyPEG-monovinylsulfone or ahydroxyPEG-monophenyl carbonate derivative) having a molecular weight offrom about 18 kDa to about 22 kDa or about 27 kDa to about 33 kDa.

A number of investigators have disclosed the preparation of linear orbranched “non-antigenic” PEG polymers and derivatives or conjugatesthereof (see, e.g., U.S. Pat. Nos. 5,428,128; 5,621,039; 5,622,986;5,643,575; 5,728,560; 5,730,990; 5,738,846; 5,811,076; 5,824,701;5,840,900; 5,880,131; 5,900,402; 5,902,588; 5,919,455; 5,951,974;5,965,119; 5,965,566; 5,969,040; 5,981,709; 6,011,042; 6,042,822;6,113,906; 6,127,355; 6,132,713; 6,177,087, and 6,180,095; see also PCTpublication WO 95/13090 and published U.S. patent application nos.2002/0052443, 2002/0061307 and 2002/0098192).

Any water-soluble mono- or bifunctional poly(alkylene oxide) having alinear or branched chain may be utilized in certain embodiments.Typically, the polyol is a poly(alkylene glycol) such as poly(ethylene)glycol (PEG). Those of skill in the art will recognize that otherpolyols, such as poly(propylene glycol) and copolymers of polyethyleneglycol and polypropylene glycol can be suitably used.

Alternatively, the chemical moiety may be joined, fused, or otherwiseattached to a target molecule by way of a naturally occurring amino acid(whether it originated in the native target molecule or was addedthrough modification).

The location of the chemical moiety in a particular target molecule mayaffect the structure and/or function of the target molecule. Forexample, if the chemical moiety is near an active binding site, themoiety may sterically block desired interactions of the protein in vivaHowever, if the chemical moiety is located far away from the activesites, it can sterically protect the target molecule from renal uptake,etc. without significantly reducing the activity of the target molecule.Likewise, if the chemical moiety is located near an antigenic epitope,it can reduce antigenicity of the target molecule in vivo. Thus, it isimportant to be able to control the location(s) at which the chemicalmoiety is joined to the target target molecule.

In certain embodiments, the non-natural amino acid does not containprimary amine or thiol side-chain groups. In some embodiments, thenon-natural amino acid is linked to a chemical moiety (such as PEG) viaa triazole linkage. The triazole linkage may be formed, for example, bycopper-mediated Huisgen [3+2] cycloaddition of an azide and an alkyne.The azide group may be provided, for example, bypara-azidophenylalanine, and the alkyne group may be provided, forexample, by an alkyne derivatized PEG reagent. In other embodiments, thealkyne may be provided by ethynyl Phenylalanine or ethynyl Trp, orhomopropargyl glycine. In still other embodiments, the azide may beprovided by azide derivatized PEG. In other embodiments, the azide maybe provided by azidohomoalanine, and the alkyne may be provided byalkyne derivatized PEG.

Historically, common chemical moieties, such as polyethylene glycol(PEG), also react with functional groups present in naturally occurringamino acids (such as the epsilon amino group in lysine or the thiolgroup in cysteine residues). Thus, these non-specific reactions resultin a final protein preparation that contains many isomers of proteinsconjugated to one or more chemical moieties at various locations withinthe protein, depending on the amino acid content of the protein. Thisrange of isomers affects the overall therapeutic effectiveness of theprotein, due to the variation of isomers contained within the finalpreparation, or requires extensive purification to obtain a singledesired isomer or isomer range. All of these requirements lead toincreased cost and effort in manufacturing proteins. While puttingprotecting groups on some amino acid residues (and subsequently removingthem) has provided some benefit, this technique also requiressignificant complications to the protein production and is largelyimpractical for manufacturing large quantities of modified proteins.

The present invention has the advantage of joining chemical moieties,including PEG, to target molecules, such as proteins, by utilizingunique functional groups in a nonnatural amino acid that can react withan activated PEG or other chemical moiety using chemical reactions thatdo not react with naturally occurring amino acids. Therefore, themethods used in the present invention provide for an efficient mode ofincorporating chemical moieties into proteins or other target moleculesat the non-natural amino acid location, which may be any desiredlocation in a protein or other target molecule. These reactions may alsobe performed in mild aqueous solutions that are not damaging to proteinsand the linkages to the chemical moieties for a stable covalent bond.These reactions may also be performed in vivo or on unpurifiedpreparations of protein, due to the lack of side reactions withbiological functional groups.

Thus, several advantages of the present methods include the ability toadd chemical moieties to the modified target molecules described hereinwhich can be conducted in aqueous buffers, in a wide range of pH, atroom temperature, and in a very short period of time.

In addition to attaching a chemical moiety, the atoms in proximity tothe functional groups could be altered, such as by adding electronwithdrawing or donating groups, or adding methyl or other groups thatadd steric hindrance to the target molecule. This can alter thereactivity of the functional groups or alter the stability of thestarting groups or the linkage formed. For example, an electronwithdrawing group such as a nitro group can be added to the phenyl ringof bromophenylalanine to increase reactivity. A cleavable linkage couldalso be placed in proximity, such as an ester or disulfide group betweenthe chemical moiety and the active group (e.g. alkyne), so that thechemical moiety could be removed from the protein slowly by hydrolysisof the ester or quickly by disulfide reduction. If necessary,interactions between sulfur atoms and the catalyst may be prevented orreduced by using excess catalyst or reversibly protecting cysteinylthiols.

Without wishing to be bound by any particular theory, PEGylation is aprocess by which oligosaccharides and synthetic polymers such aspolyethylene glycol (PEG) are site-specifically and covalently attachedto therapeutic protein target molecules. PEGylation can significantlyenhance protein half-life by shielding the polypeptide from proteolyticenzymes and increasing the apparent size of the protein, thus reducingclearance rates. Moreover, PEG conjugates can enhance protein solubilityand have beneficial effects on biodistribution. The physical andpharmacological properties of PEGylated proteins are affected by thenumber and the size of PEG chains attached to the polypeptide, thelocation of the PEG sites, and the chemistry used for PEGylation.Examples of PEG conjugation to proteins include reactions ofN-hydroxysuccinimidyl ester derivatized PEGs with lysine, 1,4-additionreactions of maleimide and vinylsulfone derivatized PEGs with cysteine,and condensation of hydrazide containing PEGs with aldehydes generatedby oxidation of glycoproteins.

PEGylation can significantly enhance protein half-life by shielding thepolypeptide from proteolytic enzymes and increasing the apparent size ofthe protein, thus reducing clearance rates. Moreover, PEG conjugates canenhance protein solubility and have beneficial effects onbiodistribution. The physical and pharmacological properties ofPEGylated proteins are affected by the number and the size of PEG chainsattached to the polypeptide, the location of the PEG sites, and thechemistry used for PEGylation. “PEG” may include target molecules of thegeneral formula CH₂CH₂O(CH₂CH₂O)_(n)CH₂CH₂. PEG includes linear polymershaving hydroxyl groups at each end other terminus, such as HO—PEG-OH.Examples of PEG conjugation to proteins include reactions ofN-hydroxysuccinimidyl ester derivatized PEGs with lysine, 1,4-additionreactions of maleimide and vinylsulfone derivatized PEGs with cysteine,and condensation of hydrazide containing PEGs with aldehydes generatedby oxidation of glycoproteins.

Some examples of PEG polymers include methoxy-PEG-OH (m-PEG), whereinone terminus is relatively inert while the other terminus is a hydroxylgroup that is subject to chemical modification. Branched PEGs may alsobe used (R—PEG-OH)_(n) in which R represents a central core moiety,including pentaerythritol, glycerol, or lysine and n represents thenumber of branching arms, which can range from three to a hundred ormore. The hydroxyl groups are further subject to chemical modification.Another branched form has a single terminus and is subject to chemicalmodification (see, for example, PCT patent application WO 96/21469).This type of PEG can be represented as (CH₃O-PEG)-_(p)R—X) where pequals 2 or 3, and R represents a central core such that lysine orglycerol and X represents a functional group such as carboxyl that issubject to chemical activation. Another branched form “pendent PEG” hasreactive groups, such as carboxyl, along the PEG backbone rather than atthe end of PEG chains. PEG-methyl maleimide, which may be used, forexample, in thiol-specific pegylation of antibodies, viruses, peptides,and proteins, aldehyde derivatives of PEG (PEG-butyraldehyde,PEG-pentaldehyde, PEG-amido-propionaldehyde,PEG-urethano-propioaldehyde) which may be used, for example, inN-terminal specific pegylation of proteins, and multi-arm PEG which areused, for example, as reactive components in hydrogel formulations.

Many PEG reagents have been developed for modifying proteins whichinvolve the covalent attachment of a PEG target molecule via theformation of a linking group between the PEG polymer and the protein.Some such reagents are unstable in the aqueous medium in which thePEGylation reaction occurs. Also, some proteins may lose in vitrobiological activity due to steric interaction with the protein's activesites upon addition of PEG.

A primary method by which site-specific pegylation of a protein may beconducted is the pegylation of a free cysteine moiety with aPEG-maleimide reagent. A PEG-sulfhydryl reactive derivative may reactwith a cysteine via a Michael addition to form a stable3-thiosuccidimidyl ether linkage. The maleimide specific sulfhydrylreagent can form a covalent bond with a cysteine residue about 1000-foldfaster than a corresponding amine, thereby selectively derivatizing thecysteine moiety. The resulting compound is very stable and cannot bereversed under physiological conditions.

Another method of enhancing protein stabilization via pegylation occursusing PEG aldehyde derivatives. This may be carried out, for example, byreacting the PEG aldehyde with a protein amine at a single site at theN-terminus of the protein, at a pH of from 5.5 to 7.5, which forms anintermediate Schiff base. If the amination process is desired at morethan one amino site on the protein, the reaction may be executed at a pHof 8.0 and above, preferably from 8.0 to 10.0. Such PEG aldehydes aretypically very stable in an aqueous medium but may be somewhat lessreactive for Schiff base formation. These reagents may be used for agreater overall selectivity for the reductive amination reaction andchoice of which protein amine is utilized for pegylation of the protein.

Copolymers of ethylene oxide and propylene oxide are closely related toPEG in their chemistry, and can be used instead of PEG in manyapplications. They have the following general formula: HO—CH₂CHRO(CH₂CHRO)_(n)CH₂CHR—OH where R is H or CH₃, CH₂CH₃, (CH₂)mCH₃.

Since PEG is water-soluble as well as soluble in many organic solvents,PEG is a useful polymer. PEG is generally non-toxic and non-immunogenic.When PEG is chemically attached to a water insoluble compound, theresulting conjugate generally becomes water soluble as well as solublein many organic solvents. Thus, as used herein, the “PEG moiety” isintended to include but not be limited to, linear and branched PEG,methoxy PEG, hydrolytically or enzymatically degradable PEG, pendentPEG, dendrimer PEG, copolymers of PEG and one or more polyols, andcopolymers of PEG and PLGA (poly(lactic/glycolic acid) of any weightand/or size.

When more than one reactive site is present in a protein (e.g., multipleamino or thiol groups) or reactive electrophiles are used, nonselectiveattachment of one or multiple PEG molecules can occur, leading to thegeneration of a heterogeneous mixture that is difficult to separate. Thelack of selectivity and positional control in the attachment of PEGchains can lead to significant losses in biological activity andpossibly enhanced immunogenicity of the conjugated protein. Modificationof proteins with amine-reactive PEGs typically results in drastic lossof biological activity due to modification of lysine residues located inregions of the protein important for biological activity. In certainsituations, bioactivity of growth hormones may be reduced 400-fold ormore. For example, bioactivity of GCSF is reduced 1,000-fold when theproteins are modified using conventional amine-PEGylation technologies(Clark et al., J. Biol. Chem. 271: 21969, 1996; Bowen et al., Exp.Hematol. 27, 425, 1999). Thus there is a need for a method that allowsfor the completely site-specific and irreversible attachment of PEGchains to molecules, including proteins.

The compositions, including proteins, comprise at least one non-naturalamino acid, e.g., a non-natural amino acid comprising a moiety where achemical moiety can be attached, or a non-natural amino acid thatincludes a chemical moiety are useful for, e.g., novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(e.g., antibodies), and e.g., the study of protein structure andfunction. (See, e.g., Dougherty, (2000) Curr. Opin. in Chem. Biol.,4:645-652, hereby incorporated by reference.)

In addition, PEG molecules (or other chemical moieties) may be attachedto non-natural amino acids through techniques other thanamine-PEGylation, thus sparing the primary amine groups of lysines fromundesirable PEGylation. The major advantages of such molecular orprotein engineering technologies include the creation ofnext-generation, proprietary pharmaceuticals that are homogeneouslymodified; retain high biological activity and remain longer in the body;have increased potency and stability and decreased immunogenicity; areconsistent lot to lot in biological activities. These techniques may beused to enhance the half-life, efficacy, and/or safety ofbio-pharmaceuticals in all areas, including the specific field ofcancer, endocrinology, infectious disease, immunology, systems medicineand inflammation, etc.

Methods of identifying incorporation of non-natural amino acids and/orchemical moieties into a target molecule are well known in the art andhave been described herein inter alia. For example some modes of testingfor incorporation of one or more chemical moiety include flowcytommetry, Northern blots, Western blots, PCR, RNA microsequencing,reporter assays, FLAG epitopes, binding to conjugate molecules (such asstreptavidin), radio-label detection, colorimetric assays, RNAseprotection assays, mass spectrometry (including MALDI and MALDI-TOF),NMR, IR, ELISA, fluorescent microscopy and any combination of these orother techniques known in the art.

Glycosylating Molecules

The invention also provides glycoproteins that comprise a saccharidemoiety and a polypeptide. In certain embodiments in the glycoproteins ofthe invention, the saccharide moiety is attached to the polypeptide by areaction product of a nucleophilic reaction between a first reactivegroup attached to an non-natural amino acid present in the polypeptideand a second reactive group attached to the saccharide moiety. Incertain embodiments, the first reactive group is an electrophilic moiety(e.g., keto moiety, aldehyde moiety, and/or the like) and the secondreactive group is a nucleophilic moiety.

A wide variety of suitable reactive groups are known to those of skillin the art. Such suitable reactive groups can include, for example,amino, hydroxyl, carboxyl, carboxylate, carbonyl, alkenyl, alkynyl,aldehyde, ester, ether (e.g., thio-ether), amide, amine, nitrile, vinyl,sulfide, sulfonyl, phosphoryl, or similarly chemically reactive groups.Additional suitable reactive groups include, but are not limited to,maleimide, N hydroxysuccinimide, sulfo-N-hydroxysuccinimide,nitrilotriacetic acid, activated hydroxyl, haloacetyl (e.g.,bromoacetyl, iodoacetyl), activated carboxyl, hydrazide, epoxy,aziridine, sulfonylchloride, trifluoromethyldiaziridine,pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone,succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone,isothiocyanate, isocyanate, imidoester, fluorobenzene, biotin andavidin.

In some embodiments, one of the reactive groups is an electrophilicmoiety, and the second reactive group is a nucleophilic moiety. Eitherthe nucleophilic moiety or the electrophilic moiety can be attached tothe side-chain of the non-natural amino acid; the corresponding group isthen attached to the saccharide moiety.

Suitable electrophilic moieties that react with nucleophilic moieties toform a covalent bond are known to those of skill in the art. In certainembodiments, such electrophilic moieties include, but are not limitedto, e.g., carbonyl group, a sulfonyl group, an aldehyde group, a ketonegroup, a hindered ester group, a thioester group, a stable imine group,an epoxide group, an aziridine group, etc.

Suitable nucleophilic moieties that can react with electrophilic moietyare known to those of skill in the art. In certain embodiments, suchnucleophiles include, for example, aliphatic or aromatic amines, such asethylenediamine. In certain embodiments, the nucleophilic moietiesinclude, but are not limited to, e.g., —NR1—NH₂ (hydrazide),—NR1(C═O)NR2NH₂ (semicarbazide), —NR1(C═S)NR2NH₂ (thiosemicarbazide),—(C═O)NR1NH₂ (carbonylhydrazide), —(C═S)NR1NH₂ (thiocarbonylhydrazide),—(SO₂)NR1NH₂ (sulfonylhydrazide), —NR1NR2(C═O)NR3NH₂ (carbazide),NR1NR2(C═S)NR3NH₂ (thiocarbazide), —O—NH₂ (hydroxylamine), and the like,where each R1, R2, and R3 is independently H, or alkyl having 1-6carbons, preferably H. In certain embodiments, the reactive group is ahydrazide, hydroxylamine, semicarbazide, carbohydrazide, asulfonylhydrazide, or the like.

The product of the reaction between the nucleophile and theelectrophilic moiety typically incorporates the atoms originally presentin the nucleophilic moiety. Typical linkages obtained by reacting thealdehydes or ketones with the nucleophilic moieties include reactionproducts such as an oxime, an amide, a hydrazone, a reduced hydrazone, acarbohydrazone, a thiocarbohydrazone, a sufonylhydrazone, asemicarbazone, a thiosemicarbazone, or similar functionality, dependingon the nucleophilic moiety used and the electrophilic moiety (e.g.,aldehyde, ketone, and/or the like) that is reacted with the nucleophilicmoiety. Linkages with carboxylic acids are typically referred to ascarbohydrazides or as hydroxamic acids. Linkages with sulfonic acids aretypically referred to as sulfonylhydrazides or N-sulfonylhydroxylamines.The resulting linkage can be subsequently stabilized by chemicalreduction.

Suitable electrophilic moieties that react with nucleophilic moieties toform a covalent bond are known to those of skill in the art. In certainembodiments, such electrophilic moieties include, but are not limitedto, e.g., carbonyl group, a sulfonyl group, an aldehyde group, a ketonegroup, a hindered ester group, a thioester group, a stable imine group,an epoxide group, an aziridine group, etc.

Suitable nucleophilic moieties that can react with electrophilic moietyare known to those of skill in the art. In certain embodiments, suchnucleophiles include, for example, aliphatic or aromatic amines, such asethylenediamine. In certain embodiments, the nucleophilic moietiesinclude, but are not limited to, e.g., —NR1—NH₂ (hydrazide),—NR1(C═O)NR2NH₂ (semicarbazide), —NR1(C═S)NR2NH₂ (thiosemicarbazide),—(C═O)NR1NH₂ (carbonylhydrazide), —(C═S)NR1NH₂ (thiocarbonylhydrazide),—(SO₂)NR1NH₂ (sulfonylhydrazide), —NR1NR2(C=O)NR3NH₂ (carbazide),NR1NR2(C═S)NR3NH₂ (thiocarbazide), —O—NH₂ (hydroxylamine), and the like,where each R1, R2, and R3 is independently H, or alkyl having 1-6carbons, preferably H. In certain embodiments, the reactive group is ahydrazide, hydroxylamine, semicarbazide, carbohydrazide, asulfonylhydrazide, or the like.

The product of the reaction between the nucleophile and theelectrophilic moiety typically incorporates the atoms originally presentin the nucleophilic moiety. Typical linkages obtained by reacting thealdehydes or ketones with the nucleophilic moieties include reactionproducts such as an oxime, an amide, a hydrazone, a reduced hydrazone, acarbohydrazone, a thiocarbohydrazone, a sufonylhydrazone, asemicarbazone, a thiosemicarbazone, or similar functionality, dependingon the nucleophilic moiety used and the electrophilic moiety (e.g.,aldehyde, ketone, and/or the like) that is reacted with the nucleophilicmoiety. Linkages with carboxylic acids are typically referred to ascarbohydrazides or as hydroxamic acids. Linkages with sulfonic acids aretypically referred to as sulfonylhydrazides or N-sulfonylhydroxylamines.The resulting linkage can be subsequently stabilized by chemicalreduction.

Other aspects of the invention include methods for synthesis of aglycoprotein by incorporating into a protein an non-natural amino acidthat comprises a saccharide moiety. A glycoprotein produced by themethod is also a feature of the invention. In certain embodiments, theincorporating step comprises using an mutant tRNA/mutant aminoacyl-tRNAsynthetase (M-tRNA/M-RS) pair, wherein the M-tRNA recognizes adegenerate codon and incorporates the non-natural amino acid thatcomprises a saccharide moiety (e.g., a β-O-GlcNAc-L-serine, atri-acetyl-β-GlcNAc-serine, a tri-O-acetyl-GaINAc-α-threonine, anα-GaINAc-L-threonine, and/or the like) into the protein in response tothe degenerate codon, and wherein the M-RS preferentially aminoacylatesthe M-tRNA with the non-natural amino acid. In one embodiment, theincorporating step is performed in vivo.

These methods can further involve contacting the saccharide moiety witha glycosyl transferase, a sugar donor moiety, and other reactantsrequired for glycosyl transferase activity for a sufficient time andunder appropriate conditions to transfer a sugar from the sugar donormoiety to the saccharide moiety. In certain embodiments, the methodfurther comprises contacting the product of the glycosyl transferasereaction with at least a second glycosyl transferase and a second sugardonor moiety. In other words, the invention provides methods in which anamino acid-linked saccharide moiety or an non-natural amino acid thatincludes a saccharide moiety is further glycosylated. Theseglycosylation steps are preferably (though not necessarily) carried outenzymatically using, for example, a glycosyltransferase, glycosidase, orother enzyme known to those of skill in the art. In some embodiments, aplurality of enzymatic steps are carried out in a single reactionmixture that contains two or more different glycosyl transferases. Forexample, one can conduct a galactosylating and a sialylating stepsimultaneously by including both sialyl transferase and galactosyltransferase in the reaction mixture.

For enzymatic saccharide syntheses that involve glycosyl transferasereactions, the recombinant cells of the invention optionally contain atleast one heterologous gene that encodes a glycosyl transferase. Manyglycosyl transferases are known, as are their polynucleotide sequences.See, e.g., “The WNW Guide To Cloned Glycosyl transferases,” (availableon the World Wide Web). Glycosyl transferase amino acid sequences andnucleotide sequences encoding glycosyl transferases from which the aminoacid sequences can be deduced are also found in various publiclyavailable databases, including GenBank, Swiss-Prot, EMBL, and others.

In certain embodiments, a glycosyl transferase of the inventionincludes, but is not limited to, e.g., a galactosyl transferase, afucosyl transferase, a glucosyl transferase, an N-acetylgalactosaminyltransferase, an N-acetylglucosaminyl transferase, a glucuronyltransferase, a sialyl transferase, a mannosyl transferase, a glucuronicacid transferase, a galacturonic acid transferase, an oligosaccharyltransferase, and the like. Suitable glycosyl transferases include thoseobtained from eukaryotes or prokaryotes.

An acceptor for the glycosyl transferases will be present on theglycoprotein to be modified by the methods of the invention. Suitableacceptors, include, for example, galactosyl acceptors such asGalβ1,4GaINAc-; Galβ1,3GaINAc-; lacto-N-tetraose-; Galβ1,3GlcNAc-;Galβ1,4GlcNAc-; Galβ1,3Ara-; Galβ1,6GlcNAc-; and Galβ1,4Glc-(lactose).Other acceptors known to those of skill in the art (see, e.g., Paulsonet al., J. Biol. Chem. 253: 5617-5624, 1978). Typically, the acceptorsform part of a saccharide moiety chain that is attached to theglycoprotein.

In one embodiment, the saccharide moiety comprises a terminal GlcNAc,the sugar donor moiety is UDP-GIcNAc and the glycosyl transferase is aβ1-4N-acetylglucosaminyl transferase. In another embodiment, thesaccharide moiety comprises a terminal GlcNAc, the sugar donor moiety isUDP-Gal and the glycosyl transferase is a β1-4-galactosyl transferase.Additional sugars can be added.

In one embodiment, the saccharide moiety comprises a terminal GlcNAc,the sugar donor moiety is UDP-GlcNAc and the glycosyl transferase is aβ1-4N-acetylglucosaminyl transferase. In another embodiment, thesaccharide moiety comprises a terminal GlcNAc, the sugar donor moiety isUDP-Gal and the glycosyl transferase is a β1-4-galactosyl transferase.Additional sugars can be added.

In one embodiment, the saccharide moiety comprises a terminal GlcNAc,the sugar donor moiety is UDP-Gal and the glycosyl transferase is aβ-1,4-galactosyl transferase.

In one embodiment, the saccharide moiety comprises a terminal GlcNAc,the sugar donor moiety is UDP-GlcNAc and the glycosyl transferase is aβ1-4N-acetylglucosaminyl transferase.

Optionally, the method further comprises contacting the product of theN-acetylglucosaminyl transferase reaction with a β1-4mannosyltransferase and GDP-mannose to form a saccharide moiety that comprisesManβ1-4GlcNAcβ1-4GlcNAc-. Optionally, the method further comprisescontacting the Manβ1-4GlcNAcβ1-4GlcNAc-moiety with an α1-3mannosyltransferase and GDP-mannose to form a saccharide moiety that comprisesManα1-3Manβ1-4GlcNAβ1-4GlcNAc-. Optionally, the method further comprisescontacting the Manα1-3Manβ1-4GlcNAcβ1-4GlcNAc- moiety with an α1-6mannosyl transferase and GDP-mannose to form a saccharide moiety thatcomprises Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-. Optionally, themethod further comprises contacting theManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-moiety with aβ1-2N-acetylglucosaminyl transferase and UDP-GlcNAc to form a saccharidemoiety that comprisesManα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-. Optionally, themethod further comprises contacting theManα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-moiety with aβ1-2N-acetylglucosaminyl transferase and UDP-GlcNAc to form a saccharidemoiety that comprisesGlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-.

The step of incorporating into a protein an non-natural amino acid thatcomprises a first reactive group, in some embodiments, comprises usingan mutant tRNA/mutant aminoacyl-tRNA synthetase (M-tRNA/M-RS) pair,where the M-tRNA preferentially recognizes a degenerate codon forwild-type tRNA, and incorporates the non-natural amino acid into theprotein in response to the degenerate codon, and wherein the M-RSpreferentially aminoacylates the M-tRNA with the non-natural amino acid.In some embodiments, the non-natural amino acid is incorporated into thepolypeptide in vivo.

The invention also provides glycoproteins that comprise a saccharidemoiety and a polypeptide. In certain embodiments in the glycoproteins ofthe invention, the saccharide moiety is attached to the polypeptide by areaction product of a nucleophilic reaction between a first reactivegroup attached to an non-natural amino acid present in the polypeptideand a second reactive group attached to the saccharide moiety. Incertain embodiments, the first reactive group is an electrophilic moiety(e.g., keto moiety, aldehyde moiety, and/or the like) and the secondreactive group is a nucleophilic moiety.

A wide variety of suitable reactive groups are known to those of skillin the art. Such suitable reactive groups can include, for example,amino, hydroxyl, carboxyl, carboxylate, carbonyl, alkenyl, alkynyl,aldehyde, ester, ether (e.g., thio-ether), amide, amine, nitrile, vinyl,sulfide, sulfonyl, phosphoryl, or similarly chemically reactive groups.Additional suitable reactive groups include, but are not limited to,maleimide, N hydroxysuccinimide, sulfo-N-hydroxysuccinimide,nitrilotriacetic acid, activated hydroxyl, haloacetyl (e.g.,bromoacetyl, iodoacetyl), activated carboxyl, hydrazide, epoxy,aziridine, sulfonylchloride, trifluoromethyldiaziridine,pyridyldisulfide, N-acyl-imidazole, imidazolecarbamate, vinylsulfone,succinimidylcarbonate, arylazide, anhydride, diazoacetate, benzophenone,isothiocyanate, isocyanate, imidoester, fluorobenzene, biotin andavidin.

The glycosylation reactions include, in addition to the appropriateglycosyl transferase and acceptor, an activated nucleotide sugar thatacts as a sugar donor for the glycosyl transferase. The reactions canalso include other ingredients that facilitate glycosyl transferaseactivity. These ingredients can include a divalent cation (e.g., Mg²⁺ orMn²⁺), materials necessary for ATP regeneration, phosphate ions, andorganic solvents. The concentrations or amounts of the various reactantsused in the processes depend upon numerous factors including reactionconditions such as temperature and pH value, and the choice and amountof acceptor saccharides to be glycosylated. The reaction medium may alsocomprise solubilizing detergents (e.g., Triton or SDS) and organicsolvents such as methanol or ethanol, if necessary.

Because the glycopolypeptides or pegylated molecules of the inventionprovide a variety of new polypeptide sequences (e.g., comprising annon-natural amino acid that comprises an amino acid, where a saccharideor PEG moiety can be linked, or an non-natural amino acid that includesa saccharide or PEG moiety, respectively in the case of proteinssynthesized in the translation systems herein, or, e.g., in the case ofthe novel synthetases, novel sequences of standard amino acids), theglycopolypeptides also provide new structural features which can berecognized, e.g., in immunological assays. Thus antibodies and antiserathat are specifically immunoreactive with an artificial polypeptide ofthe invention are also provided. In other words, the generation ofantisera, which specifically bind the polypeptides of the invention, aswell as the polypeptides which are bound by such antisera, are a featureof the invention.

The post-translational modification of proteins by glycosylation canaffect protein folding and stability, modify the intrinsic activity ofproteins, and modulate their interactions with other biomolecules. See,e.g., Varki, Glycobiology 3: 97-130, 1993. Natural glycoproteins areoften present as a population of many different glycoforms, which makesanalysis of glycan structure and the study of glycosylation effects onprotein structure and function difficult. Therefore, methods for thesynthesis of natural and non-natural homogeneously glycosylated proteinsare needed for the systematic understanding of glycan function, and forthe development of improved glycoprotein therapeutics.

Exemplary Chemistry for Addition of Chemical Moieties to Molecules

Numerous chemical moieties may be joined or linked to a particularmolecule through various known methods in the art. As an illustrativeexample, azide moieties may be useful in conjugating chemical moietiessuch as PEG or others described herein. The azide moiety serves as areactive functional group, and is absent in most naturally occurringcompounds (thus it is unreactive with the native amino acids ofnaturally occurring compounds). Azides also undergo a selective ligationwith a limited number of reaction partners, and azides are small and canbe introduced to biological samples without altering the molecular sizeof significantly.

One reaction that allows incorporation or introduction of azides tomolecules is the copper-mediated Huisgen [3+2] cycloaddition of anazide. This reaction can be used for the selective PEGylation ofproteins. (Tornoe et al., J. Org. Chem. 67: 3057, 2002; Rostovtsev etal., Angew. Chem., Int. Ed. 41: 596, 2002; and Wang et al., J. Am. Chem.Soc. 125: 3192, 2003, Speers et al., J. Am. Chem. Soc., 2003, 125, 4686;all of which are hereby incorporated by reference). The copper catalystmay be provided by ultrapure CuBr, CuSO₄ combined withtris(2-carboxyethyl) phosphine or ascorbate, by copper wire withexposure to air, or any other source. The reaction may be accelerated byaddition of a ligand such as bathophenanthrolinedisulfonic acid,tris-(triazolyl) amine, or other triazole or phosphine ligands, or bythe addition of palladium catalyst. Optionally, oxygen may be excludedfrom the reaction to improve yields. For example, Deiters et al.(Bioorg. Med. Chem. Lett. 14(23): 5743-5745, 2004) report a generallyapplicable

PEGylation methodology based on the site-specific incorporation ofpara-azidophenylalanine into proteins in yeast. The azido group was usedin a mild [3+2] cycloaddition reaction with an alkyne derivatized PEGreagent to afford selectively PEGylated protein. Also, Kiick, et al.,report incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, which does notrequire a copper catalyst but instead exploits the reaction between anazide and a phosphane to form a phospha-aza-ylide, which is then trappedby an acyl group with formation of a stable amide bond.

Reaction conditions may be varied to optimize PEGylation of anyparticular protein, such as, e.g., a modified IFNβ protein. For example,OEGylation may be performed under denaturing and/or reducing conditions.Thus, in particular embodiments, the reaction may be performed in thepresence of DTT and/or SDS. Suitable concentrations of DTT include,e.g., ranges of 0-10 mM, 0.1-10 mM, 0.1-5 mM, and 1-5 mM. In oneembodiment, the concentration of DTT is approximately 2 mM. Suitableconcentrations of SDS include, e.g., ranges of 0-6%. In one embodiments,the concentration of SDS is approximately 2%. In other embodiments,specific concentration of PEG and/or protein are utilized in thePEGylation reaction. For example, suitable concentration ranges of PEGinclude 0.1-5.0 wt % PEG, 1.0-5.0 wt % PEG, and 2.0-3.0 wt % PEG, and inone embodiment, PEG is present at approximately 2.0 wt %. Suitableconcentration of IFNβ or modified IFNβ include, e.g., 0.1-5 mg/ml and1.0-5 mg/ml. In particular embodiments, triazole is present at aconcentration of approximate 0-3-fold the Cu concentration. In furtherembodiments, a 1:1-2:1 molar ration of triazole ligand to coppercatalyst is used. In another embodiment, CuBr or CuSO4 is present at aconcentration in the range of 0.1-5.0 mM. In one specific embodiment,PEGylation of IFNβ or modified IFNβ is performed in the presence of 2 mMDTT, 3 mM triazole, 1.5 mM CuSO₄, 2% SDS, 2 wt % PEG, and 1-2 mg/ml IFN.

In other aspects of the invention, the non-natural amino acid maycontain a halogenated aryl or vinyl group (for example,para-bromophenylalanine or para-iodophenylalanine). A cross-couplingreaction may be conducted, such as a palladium-catalyzed Suzuki reactionwith PEG-phenylboronic acid, or other reaction described herein to yielda carbon-carbon linkage between the chemical moiety (such as PEG) andthe molecule. Several common procedures used historically to conjugatechemical moieties to molecules (including proteins) also react withfunctional groups present in naturally occurring amino acids, such asthe epsilon amino group in lysine or the thiol group in cysteineresidues. Thus, the non-specific reactions result in the final proteinpreparation containing many isomers of proteins conjugated to one ormore chemical moieties at different locations within the protein,depending on the amino acid sequence of the target protein.

The use of a non-natural amino acid at a particular location in a targetmolecule allows for chemical modification, such as PEGylation, to occurat that specific site. As disclosed herein, typically moleculemodification schemes utilize the chemistry of amino acid side chains toadd chemical moieties to the target molecule. In one particular example,pegylated human interferon-α-2B protein product (PEG-Intron) produces upto 14 different positions of modification, including molecules withmultiple PEGs attached. For example, the PEG-Intron results inmonopegylated positional isomers, with the PEG moiety occurring atlysine, tyrosine, histidine, serine and cysteine residues. Proteinproducts that are mixed isomers have lower activity due to the myriad oflocations where the chemical moiety is attached and since not allpositional isomers are active, or may have reduced activity.

For example, PEG-Intron has an antiviral activity of 28% of theunmodified interferon-α protein, with a range of 6-37% for individualisomer species. In addition, manufacturing costs are increased due tothe need to separate out the fraction of undesired species andadditional processing of the variable modified protein batches. Thus,there is a need in the art for production of proteins with chemicalmoieties (including PEG) that are consistently modified.

While some techniques for controlling the location of the chemicalmoiety attachment are known in the art, such as adjusting the pH of thereaction mixture, using protecting groups for some amino acid residuesduring chemical moiety conjugation, altering the folding state of theprotein to allow for better structural access to specific proteinregions, and altering the chemistry of the activated chemical moietyspecies so it is less likely to react with other nondesired functionalgroups, none of these techniques eliminates side reactions withundesired amino acid residues. One known technique avoids side reactionswith undesired amino acid residues by using protecting groups for someamino acid residues during chemical moiety conjugation, followed byremoving the protecting groups from the modified protein. However, thistechnique is cumbersome, expensive and impractical for manufacturing amodified protein product.

It is desirable to synthesize molecules, including therapeuticmolecules, in which the added chemical moiety may be specificallydirected to a target location in the molecule in order to reducevariability of the overall modified protein product and increaseactivity or other desired goal. For example, if the chemical moiety isnear an active binding site of the protein, it can sterically blockdesired interactions of the protein in vivo, if the chemical moiety islocated near an antigenic epitope, it may reduce the antigenicity of themolecule in vivo. Likewise, if the chemical moiety is located away fromactive sites, it may sterically protect the molecule from renal uptakeor clearance in vivo without reducing the activity of the molecule.

One of the advantages of certain embodiments of the present inventionincludes utilizing non-natural amino acids at specific positions wherePEGylation is desired. In certain embodiments, PEGylation chemistry canbe used that is specific to the non-natural amino acid side chain, whichresults in the PEG being added only at the desired location in thetarget molecule. The efficiency of this chemical reaction is much higherthan traditional PEGylation methods due to the absence of thecross-reactivity or other undesirable side-reactions. For example,copper catalyzed cycloaddition between an azide and an alkyne may be upto 80% efficient or greater. Such chemistry is not reactive with othercomponents of the molecule. Other, non-reactive chemistry PEGylationschemes may be utilized as well.

Since certain embodiments of the chemical reactions described hereinprovide for reactions that solely react with unique functional groups innon-natural amino acid residues, the reactions allow for naturallyoccurring amino acids to remain unmodified. For instance,palladium-catalyzed cross coupling reactions are largely unreactive withnaturally occurring amino acid residues, thus allowing for sitespecific, covalent linkage of a chemical moiety with the moleculewithout undesired conjugation elsewhere in the molecule. Anotheradvantage is that these specifically disclosed chemical reactions may beperformed in mild aqueous conditions that are not damaging to proteins.In addition, the conjugation chemistry may be reversed, such that thereactive group is present on an activated chemical moiety, rather thanthe target non-natural amino acid. Under these circumstances, theactivated chemical moiety could be reacted with nonnatural amino acidssuch as homoproparglyglycine or homoallylglycine.

In certain other embodiments, multiple different non-natural amino acidresidues may be incorporated into a target molecule and one or more ofthe non-natural amino acid residues could be conjugated to a chemicalmoiety by any of the techniques described herein.

A number of other well-known chemical reactions may be utilized toattach a chemical moiety to a protein or other molecule, some of whichare described herein. The reactive group may be either located on thetarget molecule, or on the chemical moiety selected for conjugation tothe target molecule. The Suzuki Coupling is a palladium-catalyzed crosscoupling between organobornic acid and aryl or vinyl halides,pseudo-halides (including triflates), alkyls, alkenyls and/or alkynyls.In addition, potassium trifluoroborates and organoboranes or boronateesters may be used instead of boronic salts. For more details, see forexample, Baxter, et al., J. Am. Chem. Soc., 2003, 125, 7198-7199; Wu, etal., J. Org. Chem., 2003, 68, 670-673 and Molander, et al., J. Org.Chem., 2002, 67, 8424-8429.

The Hiyama Coupling reaction may also be used to join chemical moietiesto molecules, including proteins. The Hiyama Coupling is well known inthe art and involves a palladium-catalyzed C—C bond formation betweenaryl, alkenyl, or alkyl halides or pseudohalides and organosilanes. Thesuccess of this reaction depends on the polarization of the Si—C bond,thus activation of silane with base or fluoride ions (TASF, TBAF)results in a pentavlant silicon compound. Another approach includesusing silacyclobutanes. For more details, see for example, Lee et al.,J. Am. Chem. Soc. , 2003, 125, 5616-5617; Denmark, et al., J. Am. Chem.Soc., 1999, 121, 5821-5822; Li, et al., Synthesis, 2005, 3039-3044;Murata, et al., Synthesis, 2001, 2231-2233; Lee, Org. Lett., 2000,2053-2055.

The Kumada Coupling reaction may also be used to join chemical moietiesto molecules, including proteins. The Kumada Coupling reaction is apalladium or nickel catalyzed cross coupling reaction of Grignardreagens with alkyl, vinyl or aryl halides. For more details, see forexample, Frisch, et al., Angew. Chem., 2002, 114, 4218-4221. The NegishiCoupling reaction may also be used to join chemical moieties tomolecules, including proteins. The Negishi Coupling is a nickel orpalladium catalyzed coupling of organozinc compounds with varioushalides (aryl, vinyl, benzyl or allyl). For further details, see forexample, Hadei, et al., Org. Lett., 2005, 7, 3805-3807; Huo, et al.,Org. Lett., 2003, 5, 423-425; Lutzen, et al., Eur. J. Org. Chem., 2002,2292-2297. The Stille Coupling may also be used to join chemicalmoieties to molecules, including proteins. The Stille Coupling reactionforms a C—C bond between stannanes and halides or pseudohalides. Forfurther details, see for example, Mee, et al., Angew. Chem., 2004, 116,1152-1156; Huang, et al., Tetrahedron, 2003, 59, 3635-3641; Del Valle,et al., J. Org. Chem., 1990, 55, 3019-3023; Lerebours, et al., J. Org.Chem. 2005, 70, 8601-8604.

The Heck Reaction may also be used to join chemical moieties tomolecules, including proteins. The Heck Reaction is apalladium-catalyzed C—C coupling between aryl halides or vinyl halidesand activated alkenes in the presence of a base. For further detailssee, for example, Chandrasekhar, et al., Org. Lett., 2002, 4, 4399-4401;Masllorens, et al., Org. Lett., 2003, 5, 1559-1561; Battistuzzi, et al.,Org. Lett., 2003, 5, 777-780; Mo, et al., J. Am. Chem. Soc., 2005, 127,751-760; Hansen, et al., Org. Lett., 2005, 7, 5585-5587. The FukuyamaCoupling is another reaction that may be used to join chemical moietiesto molecules, including proteins. The Fukuyama Coupling is apalladium-catalyzed coupling of organozinc compounds with thioesters toform ketones. The oxidateive addition of a thioester is followed bytransmetallation from the zinc compound. Reductive elimination leads tothe coupled product. For more details, see for example, Tokuyama, etal., J. Braz. Chem. Soc., 1998, 9, 381-387. Another reaction that may beused to join chemical moieties to molecules, including proteins, is theSonogashira Coupling. The Sonogashira Coupling reaction couples terminalalkynes with aryl or vinyl halides using a palladium catalyst, acopper(I) cocatalyst, and an amine base. For more details see, forexample, Liang, et al., J. Org. Chem., 2006, 71, 379-381; Gholap, etal., J. Org. Chem., 2005, 70, 4869-4872; Liang, et al., J. Org. Chem.2005, 70, 391-393; Elangovan, et al., Org. Lett., 2003, 5, 1841-1844;Batey, et al., Org. Lett., 2002, 1411-1414.

The Cadiot-Chodkiewicz Coupling may also be used to join chemicalmoieties to proteins or other molecules. This reaction is a copper(I)catalyzed coupling of a terminal alkyne and an alkynl halide offersaccess to unsymmetrical bisacetylenes. Further details may be found, forexample, at Marino, et al., J. Org. Chem., 2002, 67, 6841-6844. Anotherreaction that may be used to join chemical moieties to proteins or othermolecules includes the Eglinton Reaction. This reaction is an oxidativecoupling of terminal alkynes, and allows the synthesis of symmetric orcyclic bisacetylenes via reaction of the terminal alkyne with astoichiometric amount of a copper(I) salt in pyridine. In addition, theGlaser Coupling is a synthesis of symmetric or cyclic bisacetylenes viaa coupling reaction of terminal alkynes. The reaction is mechanicallysimilar to the Eglinton Reaction; the difference being the use ofcatalytic copper(I) which is reoxidized in the catalytic cycle by oxygenin the reaction medium. The Hay Coupling is a copper-catalyzed reactionthat utilizes copper-TMEDA complex. For more details on the Eglinton,Glaser, or Hay reactions, see for example, Gibtner, et al., Chem. Euro.J., 2002, 68, 408-432. Each of these references cited are herebyincorporated by reference in their entireties.

Pharmaceutical Compositions

The present invention further relates to pharmaceutical compositions andmethods of use. The pharmaceutical compositions of the present inventioninclude modified target molecules in pharmaceutical form, i.e.pharmaceutical salts, derivatives, carriers, and the like.Pharmaceutical compositions of the present disclosure may be made bymethods described herein, or other methods known in the art. In at leastone embodiment, the pharmaceutical composition exhibits at least oneimproved property selected from the group consisting of: proteinstability, protein activity, protein conformation, protein substratespecificity, protein-target binding affinity, antigen-binding ability,thermostability, protein resistance to at least one protease, proteintolerance to at least one non-aqueous environment, patient tolerance tosaid protein, increased efficacy of said protein in a patient, improveddelivery of said protein or protein product in a patient and anycombination thereof.

The present invention also relates to methods of therapeutically orprophylactically treating or diagnosing a disease or disorder byadministering a composition or agent of the present invention by anymode described herein. Such composition may be administered in vitro, invivo, ex vivo or any combination thereof.

For example, if the composition is administered ex vivo, a cell orpopulation of cells (including tissues or organs) may be obtained from asubject and contacted with an amount of a composition of the inventionthat is effective in prophylactically or therapeutically ordiagnostically effective in treating the disease, disorder or condition.Following contact with a composition of the present invention, thecells, tissues or organs may then be returned to the subject in the sameor another site.

If the composition is administered in vivo, it may be directly orindirectly administered to the cells, tissues and/or organs of asubject. For example, a particular cell or group of cells may betargeted for administration of a pharmaceutical agent or drug. Any suchmode of administration herein described may be utilized in such in vivodelivery.

Most administered protein pharmaceuticals are cleared rapidly from thebody, necessitating frequent, often daily injections. Thus, there isconsiderable interest in developing long-acting protein therapeuticsthat are able to maintain efficacious levels in the body for longperiods of time, providing patients with greater therapeutic benefits.For example, PEGylation-based drug delivery technology is a method forincreasing protein half-life.

When more than one reactive site is present in a protein (e.g., multipleamino or thiol groups) or reactive electrophiles are used, nonselectiveattachment of one or multiple PEG molecules can occur, leading to thegeneration of a heterogeneous mixture that is difficult to separate. Thelack of selectivity and positional control in the attachment of PEGchains can lead to significant losses in biological activity andpossibly enhanced immunogenicity of the conjugated protein. Modificationof proteins with amine-reactive PEGs typically results in drastic lossof biological activity due to modification of lysine residues located inregions of the protein important for biological activity. In certainsituations, bioactivity of growth hormones may be reduced 400-fold ormore. For example, bioactivity of GCSF is reduced 1,000-fold when theproteins are modified using conventional amine-PEGylation technologies(Clark et al., J. Biol. Chem. 271: 21969, 1996; Bowen et al., Exp.Hematol. 27, 425, 1999). Thus there is a need for a method that allowsfor the completely site-specific and irreversible attachment of PEGchains to molecules, including proteins.

It would be advantageous to use advanced protein engineeringtechnologies to create long-acting, “patient friendly” human proteinpharmaceuticals, by, for example, incorporating non-natural amino acidsand/or chemical moieties into a pharmaceutical drug, such that theengineered pharmaceutical may achieve longer half life and/or sustainedor even enhanced biological activity.

Multi-Drug lmmunoconjugates

Immunoconjugation may be used to increase the therapeutic efficacies ofantibodies. However, current technologies allow attachment of only asingle type of drug to an antibody. This is primarily due to thelimitations in the scope of chemistries available in the set of naturalamino acids, which do not allow precise control over theimmunoconjugation processes.

Attempts to attach multiple drugs on an antibody using currenttechnologies lead to significant heterogeneity from molecule tomolecule, and inconsistencies from lot to lot. Non-natural amino acidscan be used to provide a wide variety of new chemistries to attach drugssite-specifically, thus enabling the provision of tumor-targeted,multi-drug regimens to cancer patients. For example, the instant methodscan be used to produce immunoconjugates either by attaching a singletype of drug site-specifically on to antibodies and/or antibodyfragments to overcome issues related to heterogeneity, or by attachingmultiple drug-types site-specifically on to antibodies and/or antibodyfragments in a stoichiometrically controlled manner. In other words, themethods of the instant invention can be used to design a novel class ofimmunoconjugates that carry a combination of drugs that can be deliveredsimultaneously and specifically to a particular target site, where thetherapeutic molecules in the medicament are highly homogeneous, withlot-to-lot consistency. The major advantages of such immunoconjugatesinclude: simultaneous targeted delivery of multiple drugs that actsynergistically in treating and/or killing target cells (including tumorcells); combining drugs that act in different phases of the cell cycleto increase the number of target cells exposed to a particularpharmaceutical drug or effect; focused delivery of the pharmacologicalagent to target cells, thus maximizing the pharmaceutical benefit oreffect; minimized exposure to non-target cells, tissues or organs;precise control over drug payloads and drug ratios leading to homogenousfinal products.

In one specific example, particular cytokines (such as interferon-β) mayinhibit tumor formation, cause regression of established tumors, and/orprevent recurrence of certain cancers. See, for example, Qin, et al.P.N.A.S., V. 95, No. 24, pp. 14411-14416, (1998); Ikeda, et al.,Hepatology, 32 (2): 228-32, (2000), both of which are herebyincorporated by reference. As disclosed in the cited references,interferon β has potent antiproliferative activity against most humantumor cells in vitro, but relies on high concentrations of cytokine inorder to achieve the anti-tumor effect. Such high concentrations cannotbe utilized by parenteral protein administration because of rapidprotein clearance and systemic toxicities. Thus, a novel modifiedinterferon β that exhibits higher potentcy and sustained in vivoretention in the subject or patient, is needed in the art. In oneembodiment of the present invention, a novel, modified interferon β isprovided that fills this need.

Thus the invention provides an immunoconjugate comprising an antibody(or its functional fragment) specific for a target (e.g., a targetcell), the antibody (or fragment or functional equivalent thereof)conjugated, at specific, pre-determined positions, with two or moretherapeutic molecules, wherein each of the positions comprise annon-natural amino acid. In certain embodiments, the antibody fragmentsare F(ab′)₂, Fab′, Fab, ScFv or Fv fragments.

Immobilization of Molecules on a Solid Support

Another aspect of the invention provides a method for immobilizing oneor more target molecules, including proteins, peptides, polypeptides,biopolymers or other target molecules to a solid support including anarray, a purification column, microscopic slides, tubes, microfluidicdevices, chromatography columns or any other surface, the methodcomprising: (1) incorporating one or more non-natural amino acid(s) atspecified position(s) of the polypeptide(s) using any of the suitablemethods; (2) contacting the polypeptide(s) with a solid support toconjugate the polypeptide(s) through the non-natural amino acid(s).

In certain embodiments, the one or more target molecules are attached tothe solid support in a consistent orientation. In certain embodiments,the active site(s) of each target molecule are accessible to potentiallyinteracting target molecules. In certain embodiments, the targetmolecule of interest (or library of target molecules) is attached to asolid support through a biological or chemical linker (including any ofthe chemical moieties disclosed herein).

The solid support may comprise any known solid or semi-solid substance,including resins, glass, metals, silicon, plastics, wood, minerals,fabrics or spun fibers and any combination of these. In addition, thesolid or semi-solid support may be coated with another biological orchemical to facilitate adherence of the target molecule(s) to the solidsupport. Alternatively, such coating may be for selective adherence ofspecific target molecules or for disallowing specific target moleculesfrom adhering to the solid support.

Another aspect of the invention provides a molecular array produced byany of the suitable subject methods.

In at least one embodiment, a target molecule of the present inventionis immobilized by use of a column that has a biological or chemicalagent attached (such as a complementary amino acid tag) that selects forthe target target molecule(s). Thus, the column will selectivelyimmobilize the target molecules containing the marker through chemicalreaction. In at least one embodiment, the biological or chemical markermay be cleaved or separated from the remaining target molecule throughchemical or biological cleavage (for example, by use of enzymatic orproteolytic cleaving site).

In at least one embodiment, the one or more non-natural amino acidresidues in the modified target molecule may be used to capture theprotein on a matrix or solid support for the purpose of immobilizing thetarget molecule and/or purifying it from other proteins. In at least oneembodiment, the other proteins comprise contaminating proteins. In atleast one embodiment, the method for immobilizing a modified targetmolecule from a sample of mixed target molecules (which may containcontaminating target molecules) that includes reversibly binding themodified target molecule comprising one or more non-natural amino acidresidue to a matrix and subsequently releasing the target molecule fromthe matrix once the other target molecules in the sample have beenremoved.

Kits

The present invention further provides kits relating to any of thecompositions and/or methods described herein. Kits of the presentinvention may include methods of identifying, modifying or altering atarget molecule, as well as assays to test at least one property of themodified or altered target molecule.

For example, the kits can include one or more translation system asdescribed herein (e.g., a cell), one or more non-natural amino acid,e.g., with appropriate packaging material, containers for holding thecomponents of the kit, instructional materials for practicing themethods herein and/or the like. Similarly, products of the translationsystems (e.g., proteins such as EPO analogs comprising non-natural aminoacids) can be provided in kit form, e.g., with containers for holdingthe components of the kit, instructional materials for practicing themethods herein and/or the like.

A kit of the present invention may include devices, reagents, one ormore containers, or other components. A kit of the present invention mayalso require the use of an apparatus, instrument or device, including acomputer.

In one exemplary embodiment, naturally occurring methionine amino acidresidues are replaced by non-natural amino acids, such asazido-methionine. Since azide is a versatile functional group and isabiotic in animals as well as being resistant to oxidation andrelatively non-reactive with water. Although kinetically stable, azidesare predisposed to unique modes of reactivity owing to their largeintrinsic energy content, which has been exploited for development ofreactions, including the Staudinger ligation of azides withfunctionalized phosphines and the [3+2] cycloaddition of azides withactivated alkynes. Utilizing an auxotrophic host cell that is capable ofincorporating azidomethionine highly efficiently, the target moleculewill undergo incorporation of the non-natural amino acidazidomethionine.

For example, using an auxotrophic host cell in which phenylalaninenon-natural amino acids may be incorporated site specifically at the TTTcodon, then the target gene sequence for the target molecule will bedesigned using only a single codon of phenylalanine (TTC).

For ease in purification, the target molecule may have apoly-azidomethionine tag that would increase the rate at which thetarget molecule is able to covalently bind to the column. The tag can belinked directly to the target sequence or it may be separated from thetarget gene with a protease site, thereby enabling the user to purifythe target molecule without an azide tag.

All embodiments described herein are intended to be able to be combinedwith one or more other embodiments, even for those described underdifferent aspects of the invention.

General Techniques

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif.

(Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2002) (“Ausubel”), all of which are hereby incorporated byreference in their entireties). These texts describe mutagenesis, theuse of vectors, promoters and many other relevant topics related to,e.g., the generation of external mutant tRNA, external mutantsynthetases, and pairs thereof.

Various types of mutagenesis are used in the present invention, e.g., toproduce novel sythetases or tRNAs. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination (DNAshuffling), mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like. Additionalsuitable methods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,whether chemical or involving chimeric constructs, is also included inthe present invention. In one embodiment, mutagenesis can be guided byknown information of the naturally occurring target molecule or alteredor mutated naturally occurring target molecule, e.g., sequence, sequencecomparisons, physical properties, crystal structure or the like.

The above texts and examples found herein describe these procedures aswell as the following publications and references cited within: Sieber,et al., Nature Biotech., 19:456-460 (2001); Ling et al., Anal Biochem.254(2): 157-178 (1997); Dale et al., Methods Mol. Biol. 57:369-374(1996); I. A. Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995);W. P. C. Stemmer, Nature 370, 389-91 (1994); Arnold, Curr. Opin. inBiotech. 4:450-455 (1993); Bass et al., Science 242:240-245 (1988);Fritz et al., Nucl. Acids Res. 16: 6987-6999 (1988); Kramer et al.,Nucl. Acids Res. 16: 7207 (1988); Sakamar and Khorana, Nucl. Acids Res.14: 6361-6372 (1988); Sayers et al., Nucl. Acids Res. 16:791-802 (1988);Sayers et al., Nucl. Acids Res. 16: 803-814 (1988); Carter, Methods inEnzymol. 154: 382-403 (1987); Kramer & Fritz Methods in Enzymol.154:350-367 (1987); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel et al.,Methods in Enzymol. 154, 367-382 (1987); Zoller & Smith, Methods inEnzymol. 154:329-350 (1987); Carter, Biochem. J. 237:1-7 (1986);Eghtedarzadeh & Henikoff, Nucl. Acids Res. 14: 5115 (1986); Mandecki,Proc. Natl. Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein,Nucl. Acids Res. 14: 9679-9698 (1986); Wells et al., Phil. Trans. R.Soc. Lond. A 317: 415-423 (1986); Botstein & Shortie, Science229:1193-1201(1985); Carter et al., Nucl. Acids Res. 13: 4431-4443(1985); Grundstrom et al., Nucl. Acids Res. 13: 3305-3316 (1985);Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Smith, Ann. Rev.Genet. 19:423-462 (1985); Taylor et al., Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Wells etal., Gene 34:315-323 (1985); Kramer et al., Nucl. Acids Res. 12:9441-9456 (1984); Kramer et al., Cell 38:879-887 (1984); Nambiar et al.,Science 223: 1299-1301 (1984); Zoller & Smith, Methods in Enzymol.100:468-500 (1983); and Zoller & Smith, Nucl. Acids Res. 10:6487-6500(1982), all of which are incorporated herein by reference. Additionaldetails on many of the above methods can be found in Methods inEnzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically, for example, according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers, Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al.,Nucleic Acids Res., 12:6159-6168 (1984), or as described by Tang andTirrell J. Am. Chem. Soc. (2001) 123: 11089-11090 and Tang, et al.Angew. Chem. Int. Ed. (2001) 40:8, all of which are hereby incorporatedby reference in their entireties.

In addition, essentially any nucleic acid can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company, The Great American Gene Company, ExpressGenInc., Operon Technologies Inc. (Alameda, Calif.) and many others.

The present invention also relates to host cells and organisms for thein vivo incorporation of an non-natural amino acid via external mutanttRNA/RS pairs. Host cells are genetically engineered (e.g., transformed,transduced or transfected) with the vectors of this invention, which canbe, for example, a cloning vector or an expression vector. The vectorcan be, for example, in the form of a plasmid, a bacterium, a virus, anaked polynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation, infection by viral vectors, high velocityballistic penetration by small particles with the nucleic acid eitherwithin the matrix of small beads or particles, or on the surface.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms.

Other useful references, e.g., for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla., all of whichare hereby incorporated by reference in their entireties.

Several well-known methods of introducing target nucleic acids intobacterial cells are available, any of which can be used in the presentinvention. These include: fusion of the recipient cells with bacterialprotoplasts containing the DNA, electroporation, projectile bombardment,and infection with viral vectors, etc. Bacterial cells can be used toamplify the number of plasmids containing DNA constructs of thisinvention. For example, the bacteria are grown to log phase and theplasmids within the bacteria may be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria, (see, e.g., EasyPrep™, FlexiPrep™, both from PharmaciaBiotech; StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Theisolated and purified plasmids are then further manipulated to produceother plasmids, used to transfect cells or incorporated into relatedvectors to infect organisms.

Typical vectors contain transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular target nucleic acid.The vectors optionally comprise generic expression cassettes containingat least one independent terminator sequence; sequences permittingreplication of the cassette in eukaryotes, prokaryotes or both (e.g.,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes or both. (See, for example, Giliman & Smith,Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider,B., et al., Protein Expr. Purif. 6435: 10 (1995), all of which arehereby incorporated by reference). Additionally, a catalogue of Bacteriaand Bacteriophages useful for cloning is provided, e.g., by the ATCC,e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna etal. (eds.) published by the ATCC. Additional basic procedures forsequencing, cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY.

Non-natural amino acids may be incorporated into protein using variousmethods. For example, in one embodiment, if the non-natural amino acidis structurally/sterically similar to one of the twenty natural aminoacids, the non-natural amino acid may be incorporated into a targetprotein by way of competitive biosynthetic assimilation (see, forexample, Budisa 1995, Eur. J. Biochem 230: 788-796; Deming 1997, J.Macromol. Sci. Pure Appl. Chem A34; 2143-2150; Duewel 1997, Biochemistry36: 3404-3416; van Hest and Tirrell 1998, FEBS Lett 428(1-2): 68-70;Sharma et al., 2000, FEBS Lett 467(1): 37-40, all of which areincorporated herein by reference).

In certain embodiments, the competing natural amino acids might beselectively depleted to enhance the incorporation of non-natural aminoacids.

In another embodiment, non-natural amino acids may be incorporated intoa target molecule, including a protein, by using either a nonsensesuppressor or a frame-shift suppressor tRNA in response to amber orfour-base codons, respectively (See Bain et al., J. Am. Chem. Soc. 111:8013, 1989; Noren et al., Science 244: 182, 1989; Furter, Protein Sci.7: 419, 1998; Wang et al., Proc. Natl. Acad. Sci. U.S.A., 100: 56, 2003;Hohsaka et al., FEBS Lett. 344: 171: 1994; Kowal and Oliver, NucleicAcids Res. 25: 4685, 1997, all of which are incorporated herein byreference) Such methods insert non-canonical amino acids at codonpositions that will normally terminate wild-type peptide synthesis(e.g., a stop codon or a frame-shift mutation). These methods haveworked well for single-site insertion of novel amino acids. Thesemethods may work modestly well for multisite incorporation, if modest(20-60%) suppression efficiencies are acceptable (See Anderson et al.,J. Am. Chem. Soc. 124: 9674, 2002; Bain et al., Nature 356: 537, 1992;Hohsaka et al., Nucleic Acids Res. 29: 3646, 2001, all of which areincorporated herein by reference).

In yet another embodiment, efficient multisite incorporation may beaccomplished by replacement of natural amino acids in auxotrophicEscherichia coli strains, and by using aminoacyl-tRNA synthetases withrelaxed substrate specificity or attenuated editing activity (See, forexample, Wilson and Hatfield, Biochem. Biophys. Acta 781: 205, 1984;Kast and Hennecke, J. Mol. Biol. 222: 99, 1991; Ibba et al.,Biochemistry 33: 7107, 1994; Sharma et al., FEBS Lett. 467: 37, 2000;Tang and Tirrell, Biochemistry 41: 10635, 2002; Datta et al., J. Am.Chem. Soc. 124: 5652, 2002; Doring et al., Science 292: 501, 2001, allof which are incorporated herein by reference). This method may beuseful, particularly when it is acceptable to allow non-natural aminoacids to “share” codons with one of the natural amino acids, and whenincorporation at an unintended site does not substantially compromisethe function of the target molecule.

Examples

The following examples are provided as further illustrations and notlimitations of the present invention. The teachings of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures are hereby incorporated byreference.

Example 1 Prophetic DESIGN OF SITE-SPECIFIC PEGYLATED PROTEINS

The design of a pegylated GM-CSF, Erythropoietin (EPO), Human GrowthHormone, Phenylalanine hydroxylase, urikase, Factor VII, follitropin,G-CSF, or other target molecule may comprise a multi-step process. Inthe case of EPO, which wild type sequence contains two methionine aminoacids—including one at the amino terminus, only one methionine wouldrequire substitution. In the case of G-CSF, the wild type sequence doesnot contain any arginine residues. Thus, an arginine residue could beintroduced at any desirable location in the molecule and subsequentlysubstituted or replaced with a non-natural amino acid. Likewise, forHuman Growth Hormone, the wild type sequence only contains a singletryptophan residue, phenylalanine hydroxylase contains only 3 methionineresidues and 3 tryptophan residues, and follitropin contains only 5methionine residues.

In an optional first step, existing specific target wild type aminoacids (for example, methionine residues) will be designed to othernaturally occurring amino acid residues. The amino acid residues thatreplace the target wild type amino acids would likely support themolecule's native structural stability and/or activity. Next, specificamino acid residue positions will be selected for incorporation of oneor more non-natural amino acid. The selected amino acid residuepositions for incorporation of the non-natural amino acids may be thesame amino acid residues that were replaced by other naturally occurringamino acid residues in the optional first step, or may be naturallyoccurring amino acid residues that were not changed, or may be stillother positions corresponding to codons in the nucleotide sequence noteffectively encoding any natural amino acid including, for example, stopcodons, 4 or 5 base codons, or bias codons. The non-natural amino acidresidues may or may not be a corresponding analog to the specific aminoacid being replaced in the optional first step.

Replacement of amino acid residues with other naturally occurring aminoacid residues and/or incorporation of non-natural amino acid residuesmay be accomplished by any methods known or as-yet unknown in the art.For example, amino acid specific external mutant tRNA synthetase-tRNApairs may be employed to increase the yield and efficiency of thesubstitution (including, for example, stop codons such as amber codon,ochre codon, or opal codon; degenerate codons such as wobble codons,bias codons, 4 or 5 base pair codons, sixth box codons, or other means)or other codons which typically specifiy a naturally occurring aminoacid but is distinct from the other codons used in the protein to encodethat particular naturally occurring amino acid. Host cell lines thathave been engineered to preferentially incorporate a particular aminoacid (or amino acids) may be utilized, including but not limited toauxotrophic host cell lines. The host cell line may be modified by sitedirected mutagenesis (including, for example, by PCR, restrictiondigests and re-ligation, chemical mutagenesis, or other means). Othermethods of altering a particular amino acid residue may be used, such asengineering host cells with exogenous or external mutant AARS with orwithout a cognate tRNA, to facilitate incorporation of a particularnon-natural amino acid.

In the next step, a chemical moiety (such as polyethylene glycol) isadded to the non-natural amino acid residue in the molecule, therebyforming a pegylated GM-CSF molecule.

The amino acid residues selected for replacement by naturally occurringamino acid residues and/or non-natural amino acid residues may bedetermined, in part, by evaluating energy calculations and/orthree-dimensional structural location of the residues. Additionally,replacement amino acids may be selected by alignment of nucleic acid oramino acid sequences of related genes or proteins, respectively. Suchsequences may be from the same species or different species.

Optionally, rather than replacing all specific target wild type aminoacid residues with other naturally occurring amino acid residues, asdescribed in the optional first step, an alternative approach may beused. For example, one or more specific target wild type amino acidresidue(s) may be retained in the molecule, which may then besubstituted with a non-naturally occurring amino acid residue and thesubsequent addition of a chemical moiety made to the non-naturallyoccurring amino acid.

Example 2 Prophetic SITE-SPECIFIC PEGYLATION OF GM-CSF

A GM-CSF molecule contains four wild type methionine amino acid residuesat positions 36, 46, 79 and 80. There are at least two possibilities forinserting a site-specific methionine analog into GM-CSF for use as ananchoring residue for pegylation.

One option would be to retain one of the four methionine residues in theGM-CSF molecule and replace the three other methionine residues withother naturally occurring amino acid residues. Selecting which threemethionine residues will be replaced and/or selecting which naturallyoccurring amino acid residues shall replace the three wild typemethionine residues may be determined, in part, by evaluating energycalculations as described herein. Additionally, replacement amino acidsmay be selected by alignment of nucleic acid or amino acid sequences ofrelated genes or proteins, respectively. The sequences may be from thesame species or different species.

A second option would be to replace all four methionine residues in theGM-CSF molecule and add a methionine residue at another specificlocation on the molecule. Next, the added or retained methionine residuewill be replaced with a non-natural amino acid residue. Again, selectingwhether three or four methionine residues are replaced, as well asselecting the specific location of the newly added methionine residuemay be determined, in part, by evaluating energy calculations andalignments of related sequences.

Example 3 Prophetic ENERGY CALCULATIONS FOR SITE-SPECIFIC PEGYLATEDGM-CSF

Energy calculations for the target molecule discussed in the previousExample may be conducted by any known method, some of which aredescribed herein. The sequence and number of energy calculations may beperformed in a number of ways. For example, a point mutation calculationmay be performed for each selected methionine position (which includepositions 36, 46, 79 and 80). Alternatively or additionally, combinationmutation calculations may be performed for all four methionines suchthat one methionine is retained in its wild type position, while theother three methionine residues will be varied simultaneously to othernaturally occurring amino acids. In this manner, it may be determinedwhether all four methionine residues will be replaced with other aminoacid residues, or if one methionine residue will be retained while theother three are replaced with other naturally occurring amino acidresidues.

In order to limit energy calculations, the structural architecture ofthe molecule may be considered. For example, replacing the wild typemethionine residues in the core of the GM-CSF molecule may be restrictedto only hydrophobic amino acids, in order to maintain the structuralintegrity of the molecule. Whereas methionine residues that are locatedat positions that are partially or completely solvent exposed may bereplaced with a broader selection of amino acid residues.

Once energy calculations for replacing the wild type target amino acidresidue(s) (e.g. methionine residues) have been conducted, the mostenergetically favorable model GM-CSF molecule(s) will be generated andtested for stability and function. Modified GM-CSF molecules that testsuccessfully for stability and function may be used for further designof insertion and/or replacement of methionine residues with non-naturalamino acid residues.

In addition to energy calculations, determining the positions forincorporating non-natural amino acid residues (e.g. methionine analogs)will be based on the overall structure and architecture of the GM-CSFmolecule. For example, favorable positions for inserting or replacing amethionine residue with a non-natural amino acid residue may includesurface-exposed positions, preferably distal from the receptor-bindingsite. Positions to avoid may possibly include core amino acid residuesand/or residue positions at the dimer interface or that areunsymmetrical with regard to the dimer, as well as amino acid positionsthat are highly conserved (such as surface amino acids) residues.

Example 4 Prophetic SEELCTION OF NON-NATURAL AMINO ACIDS

The selection of non-natural amino acid residues for replacement of thetarget amino acid residue (whether retained in the wild type position oradded after replacement of all specific target amino acid residues) mayinclude choosing any known or newly generated non-natural amino acidthat is capable of retaining the protein's structure and/or function orcapable of being utilized by the endogenous protein translationalapparatus of the host cell. In order to preserve the structuralintegrity of the GM-CSF molecule, the non-natural amino acid residue maybe an analog of the target wild type amino acid residue. For example, amethionine residue may be replaced in the GM-CSF molecule with amethionine analog, such as homoproparglyglycine (HPG) orazidohomoalanine (AHA). Such a substitution may occur in a methionineauxotrophic cell line, or may utilize an overexpressed methionyl-tRNAsynthetase, or a mutant aminoacyl-tRNA synthetase capable ofincorporating the non-natural amino acid at the methionine position.

Determining which non-natural amino acid residues to incorporate intothe GM-CSF molecule may be conducted, in part, by evaluating energycalculations. For example, using an existing (or synthesizing a new)rotamer library for the non-natural amino acids may be used. The rotamerlibrary may be based on the torsional angles of other known methionineanalogs, if exact rotamers of HPG and/or AHA are not known. Once therotamer library is obtained, point mutation calculations may beperformed as described herein, in order to determine which non-naturalamino acid replacement is most energetically favorable.

Example 5 Prophetic SITE-SPECIFIC MODIFICATION OF OTHER PROTEINS

The aforementioned steps and Examples may also apply to other molecules,such as interferon-α, interferon-β, Factor VII, hematopoietic growthfactors, monoclonal antibodies (including bispecific antibodies andtoxins), insulin-like growth factor I, insulin, human Growth Hormone,IL-2, erythropoietin, G-CSF, GM-CSF, Phenylalanine hydroxylase,prolactin, luteinizing hormone, follitropin, parathyroid hormone,proglucagon, glucagon, GLP-1, GLP-2, GLP receptors, exendins (includingexendin-4), exendin receptors, or thrombolytics. Additionally, thespecific target wild type amino acid may vary and may include anynaturally occurring amino acid, including alanine, arginine,asparagines, aspartic acid, glutamine, glutamic acid, glycine,methionine, proline, serine, leucine, cysteine, tryptophan, isoleucine,phenylalanine, tyrosine, threonine, histidine, valine, and lysine. Thenon-natural amino acids may contain functionally important side chains,such as alkyne and azide derivatives of the particular amino acids.

Example 6 Prophetic METHODS OF SITE-SPECIFIC PEGYLATION

Proteins or other molecules may have a chemical moiety, includingpolyethylene glycol, attached or fushed to a particular amino acid, andespecially a non-natural amino acid residue as described here in otherareas of the application. The protein containing the non-natural aminoacid (which may include a halogenated aryl or vinyl group, such aspara-bromophenylalanine or para-iodophenylalanine) may then be pegylatedvia a conjugation reaction that is otherwise unreactive with theendogenous and naturally occurring amino acids of the proteins. Suchconjugation reactions are known in the art and includepalladium-catalyzed Suzuki reaction with PEG-phenylboronic acid,palladium-catalyzed Sonogashira coupling with PEG-alkyne,palladium-catalyzed Heck coupling with PEG-alkene or palladium-catalyzedHiyama reaction with PEG-silane. These palladium-catalyzed reactions, aswell as others, are described in detail in other areas of theapplication. These reactions result in a carbon-carbon linkage betweenthe PEG and the target protein.

In other aspects of the invention, the non-natural amino acid maycontain a halogenated aryl or vinyl group (for example,para-bromophenylalanine or para-iodophenylalanine). A cross-couplingreaction may be conducted, such as a palladium-catalyzed Suzuki reactionwith PEG-phenylboronic acid, or other reaction described herein to yielda carbon-carbon linkage between the chemical moiety (such as PEG) andthe molecule. Several common procedures used historically to conjugatechemical moieties to molecules (including proteins) also react withfunctional groups present in naturally occurring amino acids, such asthe epsilon amino group in lysine or the thiol group in cysteineresidues. Thus, these non-specific reactions result in the final proteinpreparation containing many isomers of proteins conjugated to one ormore chemical moieties at different locations within the protein,depending on the amino acid sequence of the target protein.

In one particular example, pegylated human interferon-α-2B proteinproduct (PEG-Intron) includes up to 14 monopegylated and multipegylatedpositional isomers, with the PEG moiety occurring at lysine, tyrosine,histidine, serine and cysteine residues. Protein products that are mixedisomers may have lower activity due to the myriad of locations where thechemical moiety is attached. For example, PEG-Intron has an antiviralactivity of 28% of the unmodified interferon-α protein, with a range of6-37% for individual isomer species. In addition, manufacturing costsare increased due to the need to separate out the fraction of undesiredspecies and additional processing of the variable modified proteinbatches. Thus, there is a need in the art for production of proteinswith chemical moieties (including PEG) that are consistently modified atspecific preferred sites.

While some techniques for biasing the location of the chemical moietyattachment are known in the art, such as adjusting the pH of thereaction mixture, using protecting groups for some amino acid residuesduring chemical moiety conjugation, altering the folding state of theprotein to allow for better structural access to specific proteinregions, and altering the chemistry of the activated chemical moietyspecies so it is less likely to react with other nondesired functionalgroups, none of these techniques eliminates side reactions withundesired amino acid residues. One known technique avoids side reactionswith undesired amino acid residues by using protecting groups for someamino acid residues during chemical moiety conjugation, followed byremoving the protecting groups from the modified protein. However, thistechnique is cumbersome, expensive and impractical for manufacturing amodified protein product, and requires that the protein be synthesizedby chemical means rather than by fermentation.

It is desirable to synthesize molecules, including therapeuticmolecules, in which the added chemical moiety may be specificallydirected to a target location in the molecule in order to reducevariability of the final modified protein product and increase activityor other desired goal. For example, if the chemical moiety is near anactive binding site of the protein, it can sterically block desiredinteractions of the protein in vivo, if the chemical moiety is locatednear an antigenic epitope, it may reduce the antigenicity of themolecule in vivo. Likewise, if the chemical moiety is located away fromactive sites, it may sterically protect the molecule from renal uptakeor clearance in vivo without reducing the activity of the molecule.

Since certain embodiments of the chemical reactions described hereinprovide for reactions that solely react with unique functional groups innon-natural amino acid residues, the reactions allow for naturallyoccurring amino acids to remain unmodified. For instance,palladium-catalyzed cross coupling reactions are largely unreactive withnaturally occurring amino acid residues, thus allowing for sitespecific, covalent linkage of a chemical moiety with the moleculewithout undesired conjugation elsewhere in the molecule. Anotheradvantage is that these specifically disclosed chemical reactions may beperformed in mild aqueous conditions that are not damaging to proteins.In addition, the conjugation chemistry may be reversed. For example,homoproparglyglycine could be coupled with a bromophenyl-PEG by aSonogashira coupling. Thus, in some embodiments, the reactive group ispresent on an activated chemical moiety, rather than the targetnon-natural amino acid.

In certain other embodiments, multiple different non-natural amino acidresidues may be incorporated into a target molecule and one or more ofthe non-natural amino acid residues could be conjugated to a chemicalmoiety by any of the techniques described herein.

A number of other well-known chemical reactions may be utilized toattach a chemical moiety to a protein or other molecule, some of whichare described herein. The reactive group may be either located on thetarget molecule, or in a bifunctional linker group that reacts with thenon-natural amino acid and with the chemical moiety to be attached. TheSuzuki Coupling is a palladium-catalyzed cross coupling betweenorganobornic acid and aryl or vinyl halides, pseudo-halides (includingtriflates), alkyls, alkenyls and/or alkynyls. In addition, potassiumtrifluoroborates and organoboranes or boronate esters may be usedinstead of boronic salts. For more details, see for example, Baxter, etal., J. Am. Chem. Soc., 2003, 125, 7198-7199; Wu, et al., J. Org. Chem.,2003, 68, 670-673 and Molander, et al., J. Org. Chem., 2002, 67,8424-8429.

For a Sonogashira Coupling, PEG-alkyne can be synthesized by reactingpropargylamine with monomethoxy-poly (ethylene glycol)-NHS, where NHS isany N-Hydroxysuccinimidyl ester of PEG designed for reaction withamines. PEG-alkyne can also be synthesized by reductive aminationbetween monomethoxy-poly(ethylene glycol)-aldehyde and propargylaminewith a reducing agent, such as sodium cyanoborohydride. The PEG-alkynecan then be conjugated to the protein containing p-bromophenylalanine inits sequence.

For a Heck Coupling, PEG-alkene can be synthesized by reactingallylamine with an activated PEG as described above for a SonogashiraCoupling.

A general palladium catalyzed reaction may use Pd(Oac)2, Na₂DCl₄ orPdCl₂, for example. A ligand, such as tris(3-sulfonato-phenyl)phosphinetrisodium, 2-(di-tert-butylphosphino)ethyltrimethylammonium chloride, orphenylbis(3-(N,N-Dimethylguanidino)phenyl)phosphine dihydrochloride maybe added to accelerate the reaction. A base such as triethylamine,pyrrolidine, Na₂CO₃, diisopropylamine or tetrabutylammonium acetate maybe added to accelerate the reaction, although it may also occur inaqueous buffer solutions with acidic pH. In the case of a Sonogashirareaction, a copper co-catalyst such as Cu(I) is added. The activated PEGspecies and the aryl halide-containing non-natural amino acid speciesare combined with the above reagents in water to couple the two speciestogether.

These reactions may proceed in aqueous solutions at a lower temperaturesuch as 4° C., room temperature, 37° C., or elevated temperatures.Exclusion of oxygen may aid the kinetics but is not necessary. Iodinatedaryl groups are more active but brominated aryl groups may also be used.The addition of an electron-withdrawing group to the phenyl ring, suchas a nitro or acetyl group, may improve reactivity, especially for thebrominated species. This reaction is beneficial in that the reactantsand catalysts undergo few, if any, side reactions with naturallyoccurring amino acids. These reactions also provide site-specificconjugation of PEG to non-natural amino acid residues incorporated intothe molecule. The C—C bond (single, double or triple bond) formed inthis conjugation reaction between PEG and the non-natural amino acid isstable, both in storage conditions and in vivo.

Example 7 Prophetic COPPER CATALYZED SITE-SPECIFIC PEGYLATION

In another example, a PEG-alkyne may be conjugated to azidohomoalaninevia a [3+2] copper catalyzed cycloaddition to yield a triazole linkagebetween the PEG and the protein. In this reaction, the copper catalystmay be provided by ultrapure CuBr, by CuSO₄ combined with a reducingagent such as tris(2-carboxyethyl) phosphine, ascorbate, ordithiothreitol, by copper wire with exposure to air, or other sources.In addition, the reaction may be further accelerated by adding a ligand,such as bathophenanthrolinedisulfonic acid, tris-(triazolyl)amine, orother triazole or phosphine ligands, or by adding palladium catalysts.Futhermore, the degree of exposure to oxygen or the redox state of thereaction may be controlled to improve reaction yields.

Example 8 Prophetic CODON-MODIFIED GENES

In another example, a gene for a target molecule (such as a protein)will be designed using only a single codon of a target amino acid, suchas phenylalanine (TTC), and a tag will be added to the target moleculecontaining the TTT wobble phenylalanine codon. The non-natural aminoacid joined with the reactive chemical moiety (the non-naturalphenylalanine, in this case), will be incorporated only at the tagregion using a tRNA-Phe (outfitted with the AAA anticodon) designed toincorporate the non-natural phenylalanine analog at the wobble TTTcodon. The molecule or protein may be bound to a column containing achemical group reactive with the side chain of the non-naturalphenylalanine analog contained specifically in the protein tag region,and may optionally contain a protease or other enzymatic cleavage site.The amino acid tag and/or cleavage site may be located on either end ofthe molecule (i.e. the N-terminal or C-terminal end). The amino acid tagmay be linked directly to the molecule or protein sequence, and the tagmay be separated from the rest of the molecule by a protease or othercleavage site.

For example, any of the following molecules may be constructed bymethods known in the art, including mutating tRNA from eukaryotic orprokaryotic sources to be outfitted with the AAA anticodon (tRNA^(Phe)_(AAA)) which reads UUU codons faster than wild-type tRNA^(Phe) _(GAA).The tRNA^(Phe) _(AAA) is then selectively charged with an non-naturalamino acid and multiple site-specific incorporation of the analog in theprotein tag results. (For more details see, for example, Kwon, et al.,J. Am. Chem. Soc. 2003, 125, 7512-7513):

-   1. START codon—(TTT)_(N)—Protease Site—Target Protein/Molecule-   2. START codon—(TTT)_(N)—Target Protein/Molecule-   3. START codon—Target Protein/Molecule—(TTT)_(N)-   4. START codon—Target Protein/Molecule—Protease Site—(TTT)_(N)

Example 9 Prophetic EXPRESSION OF SITE-SPECIFIC MODIFIED PROTEINS

While any phenylalanine analog may be used in this Example, an E. coliauxotrophic strain with a mutant yeast phenylalanine tRNA synthetaseand/or modified yeast tRNA is capable of incorporating phenylalanineanalogs (such as azido-phenylalanine, alkyne phenylalanine oracetyl-phenylalanine) at specific wobble codons with littlemisincorporation of the analog in the target protein/molecule at otherphenylalanine positions.

Misincorporation of natural phenylalanine into the tag region (if a tagis included) may be controlled by providing the cells with asignificantly higher concentration of the phenylalanine analog comparedto naturally occurring phenylalanine residues in the medium. Since alower concentration of phenyalanine may limit the overall protein yield,the poly(TTT) tag may also be increased in length.

Finally, the poly(TTT) tagged molecules may be immobilized on a solidsupport surface by preparation of a column or other surface containingthe corresponding amino acid.

Example 10 EXPRESSION VECTOR FOR MODIFIED INFβ

An E. coli synthetic gene encoding a 20 kDa modified human interferon-βprotein with a single methionine codon (at the amino terminus) wasamplified by polymerase chain reaction (PCR) using overlappingoligonucleotides (kit from OPERON®). The synthetic gene was cloned intothe pQE30 expression vector (available from QIAGEN®) under the controlof a T5-lac-lac promoter/regulator using standard molecular biologytechniques, thus forming an interferon-β mutein.

Example 11 MODIFIED INFβ

Different penultimate amino acid residues were introduced into themutein by standard molecular biology site-directed mutagenesis. Theoligonucleotide sequences encoding the N-terminal tryptic peptide of 11amino acids are listed in the Table 1 below. The sequences of all of thecloned genes were confirmed by DNA sequencing, using standard methods.

TABLE 1 NUCLEIC ACID AND AMINO ACID SEQUENCES Name N-terminaloligonucleotide sequence Peptide Sequence IFNβ-2AAtggcgtataatctgttaggctttctgcaacgt XQYNLLGFLQR (SEQ ID NO: 7) (SEQ ID NO:8) IFNβ-2S Atgagctataatctgttaggctttctgcaacgt XSYNLLGFLQR (SEQ ID NO: 9)(SEQ ID NO: 10) IFNβ-2G Atgggctataatctgttaggctttctgcaacgt XGYNLLGFLQR(SEQ ID NO: 11) (SEQ ID NO: 12) IFNβ-2HAtgcactataatctgttaggctttctgcaacgt XHYNLLGFLQR (SEQ ID NO: 13) (SEQ IDNO: 14) IFNβ-2Q Atgcagtataatctgttaggctttctgcaacgt XQYNLLGFLQR (SEQ IDNO: 15) (SEQ ID NO: 16) IFNβ-2E AtggagtataatctgttaggctttctgcaacgtXEYNLLGFLQR (SEQ ID NO: 17) (SEQ ID NO: 18) CALCULATED PEPTIDE MOLECULARWEIGHT (Daltons) Name X = Met X = AHA X = HPG X Cleaved IFNβ-2A 1324.701319.62 1302.63 1193.66 IFNβ-2S 1340.69 1335.61 1318.62 1209.65 IFNβ-2G1340.68 1305.60 1288.61 1179.64 IFNβ-2H 1390.72 1385.64 1368.65 1259.68IFNβ-2Q 1381.72 1376.64 1359.65 1259.68 IFNβ-2E 1382.70 1377.62 1360.631251.66

Example 12 EXPRESSION OF MODIFIED INFβs

The pQE30 expression vector containing the synthetic interferon-β genewas transformed with a helper plasmid (pREP4 from QIAGEN®) into amethionine auxotrophic host cell (M15MA) (Link, Tirrell, J. Am. Chem.Soc. 125: 11164-11165 (2003)). Two antibiotics (100 mg/L carbenicillinand 50 mg/L kanamycin) were used in all culture media for selection ofboth pQE30 and pREP4 plasmids.

A single colony was selected and used to inoculate LB broth forovernight growth at 37° C. The overnight culture was diluted 50 fold thenext morning into fresh LB media, and the cells were allowed to grow at37° C. until the concentration was approximately 1 (OD=600). The culturewas then centrifuged to obtain a cell pellet and remove the LB media.Cells were resuspended in M9 minimal media and grown at 37° C. for halfan hour. Cells were centrifuged again, and resuspended in M9 minimalmedia supplemented with 19 amino acids (no methionine). The cell culturewas supplemented with 50 mg/L of L-azidohomoalanine (AHA) (MEDCHEM®, WA)or L-homoproparglyglycine (HPG) (Tirrell Lab, CalTech). Parallelcultures with and without 25 mg/L methionine were grown as controls. Afinal concentration of 1 mM IPTG was added last to induce recombinantprotein expression (via inducible promoter). Cells were harvested 2hours post induction.

Example 13 ANALYSIS OF RECOMBINANT PROTEINS

Recombinant proteins were analyzed by matrix assisted laserdesorption/ionization mass spectrometry (MALDI-MS). First, recombinantmuteins were separated from endogenous E. coli proteins by 4-20%SDS-PAGE under reducing conditions, using standard technics. Theinterferon-β mutein band was visualized by Coomassie blue stain orSureBlue Safestain (INVITROGEN®), and was excised from the gel andsubjected to overnight trypsin digestion at 37° C. after destaining andmodification with iodoacetamide. Following sample drying, it wasre-dissolved in 0.1% trifluoroacetic acid (TFA) containing 2%acetonitrile. The same was then desalted by using wall-coated C18micropipette tips (NEW OBJECTIVE®) and eluted in 10-20 microliters of60% acetonitrile with 0.1% TFA. The eluted sample was mixed sith anequal volume of 10 mg/mL alpha-cyano-4-hydroxycinnamic acid in 70%acetonitrile containing 0.1% TFA plus 5 mM ammonium dihyrogen phosphate(ALDRICH®). One microliter was spotted on an OPTI-TOF® 96 well insert(APPLIED BIOSYSTEMS®) and analyzed using a 4800 MALDI TOF/TOF analyzercalibrated for a mass range of 900 to 4000 Da with “4700 calibrationmix” (APPLIED BIOSYSTEMS®).

For mass spectrometry data acquisition, 100 laser shots were fired at 20different random locations on the sample spot (total of 2000 laser shotsper sample). For tandem mass spectrometry (MSMS) data acquisition, up to3000 laser shots were accumulated per precursor ion. The N-terminalamino acid residues were confirmed by the presence of anticipatedfragment ions in their respective tandem mass spectra.

Example 14 PROCESSING OF N-TERMINAL UNNATURAL AMINO ACIDS IN RECOMBINANTPROTEINS IN E. COLI

We demonstrate the effects of the penultimate amino acid residue (theamino acid residue directly following the initiator methionine) on theprocessing of two non-natural amino acids, L-azidohomoalanine (AHA) andL-homoproparglyglycine (HPG) at the amino terminus of proteins in E.coli. We have identified several specific amino acids at the penultimateposition that can be used to efficiently retain or remove the aminoterminal AHA or HPG.

Recombinant interferon-β mutein was isolated by washing the host cellinclusion bodies, followed by separation via 4-20% SDS-PAGE. Aftertransferring the product to a PVDF membrane, the interferon-β band wascut and analyzed with five cycles of Edman degradation on a sequencermachine equipped with on-line HPLC system. Routinely, 1.0 pmolPTH-standards were used for calibration. S4 solvent, which transfers thePTH-derivatives to HPLC, contains 1.2 pmol PTH-norvaline thus acting asan internal calibrant to independently monitor transfer to the HPLC.

Free non-natural amino acids (HPG, AHA, 2,4-diaminobutyric acid) weresubjected to N-terminal sequencing to establish their elution time andstability to the sequencing conditions. A synthetic peptide containingAHA at the N-terminus (X-SYNLLG, where X=AHA) was custom synthesized byMEDCHEM® (Federal Way, Wash.). X-SYNLLG was used as a standard togenerate a correlation factor to convert the AHA peak area to its molaramount. The percentage of cleaved product was calculated by dividing theamount of protein initiated at the second position by the sum amount ofprotein initiated at both the first and second positions. The efficiencyof cleavage is reported as the mean values of 2-4 sequence cycles.Percentage of amino-terminal processed proteins based on amino-terminalsequencing analysis are presented in TABLE 2 below.

TABLE 2 Percentage Cleaved Product Name with AHA with HPG IFNβ-2A 96 91IFNβ-2S 80 80 IFNβ-2G 52 33 IFNβ-2H 8 0 IFNβ-2Q 0 0 IFNβ-2E 0 0

Thus, the extent of processing of AHA or HPG at the N-terminus dependson the identity of the penulatimate amino acid residue. Of the threeamino acids that favor the removal of N-terminal methionine (alanine,glycine and serine), alanine is most efficient (90-100%). Therefore,potentially all penulatimate residues that are inactive for methinonineAP cleavage of N-terminal methionine will also retain N-terminal AHA orHPG, as shown for histidine, glutamine, and glutamic acid. Furthermore,manipulating MetAP expression levels or substrate binding site mayrepresent another strategy for desired processing of N-terminal UAAs.

Example 15 MODIFICATION OF HUMAN IFNβ

A human interferon-β molecule was modified according to the methodsdescribed herein. The amino acid residues at positions 1 (methionine), 2(serine), 17 (cysteine), 36 (methionine), 40 (isoluecine), 44(isoleucine), 62 (methionine), and 117 (methionine) were substituted toother natural or non-natural amino acids. In particular, the amino acidat residue position 1 (methionine) was substituted to eitherazidohomoalanine or homoproparglycine. The amino acid at position 2(serine) was substituted to alanine, glycine, histidine, glutamine, orglutamic acid. The amino acid residue at position 36 (methionine) wassubstituted to threonine, alanine or isoleucine. The amino acid residueat position 40 (isoleucine) was substituted to phenylananine or leucine.The amino acid residue at position 44 (isoleucine) was substituted toleucine. The amino acid residue at position 62 (methionine) wassubstituted to leucine, isoleucine, valine, glutamine, serine,threonine, histidine, asparagines, tyrosine, phenylalanine, alanine, orglycine. The amino acid residue at position 117 (methionine) wassubstituted to threonine, tyrosine, serine or glycine. The resultingmodified human interferon beta molecule produced a stably folded proteinwith functional activity.

The particular amino acid incorporated was chosen based on a number ofcriteria, including sequence comparison of the human interferon-β genewith those from other species. A mutant interferon-β retained gene andprotein function when the methionine residue at amino acid position 36was replaced with threonine, alanine, or isoleucine, as well as when theserine at position 2 was replaced with either serine, alanine,histidine, glycine, glutamine (preferred) or glutamic acid. Otherinterferon-β mutants were synthesized with retained gene and proteinfunction when the methionine residue at amino acid position 117 wasreplaced with threonine, tyrosine, serine, or glycine.

Example 16 ANALYSIS OF IFNβ MUTEINS

When the methionine residue at amino acid position 62 of the humaninterferon-β sequence was replaced with any single naturally occurringamino acid residue, including leucine, isoleucine, valine, glutamine,serine, threonine, histidine, asparagines, tyrosine, phenylalanine,alanine, or glycine, further mutations were needed for function and/orstability.

Thus, the isoleucine residue at amino acid position 40 and/or theisoleucine residue at amino acid position 44 were also substituted withother amino acid residues, since residues at these positions werepredicted to interact with the residue at amino acid position 62.

Sequence analysis indicated the sequence of Gallus gallus interferon-βcontained an isoleucine residue at amino acid position 62, aphenylalanine residue at amino acid position 40, combined with a leucineresidue at amino acid position 44 (“chicken triple”). According to thecrystal structure and as predicted by the computational modeling, theamino acid residues at positions 40 and 44 form a non-covalent bond orotherwise interact with the amino acid at position 62 of theinterferon-13 molecule (See Tables 3-5). The corresponding substitutionswere made in the human interferon-β mutants and the resultingmulti-substituted mutant exhibited increased activity (see Figures).

By comparison, substituting the methionine residue at position 62 with aleucine residue, combined with substituting the isoluecine residue atposition 40 with a leucine residue, corresponding to the Australianechidna species sequence, failed to produce a stably folded orfunctional protein.

TABLE 3 COMPUTATIONAL PREDICTIONS OF Met62 WITH ASSOCIATED RESIDUES, 40AND 44 62 40 44 Total Energy M I I −22.91406 Human T I I −15.94224 I I I−10.97000 F I I −17.28953 L I I −9.61812 I F L −22.81071 Chicken L L I−5.78861

TABLE 4 REPEAT BIOASSAY OF Met SUBSTITUTIONS APPROXIMATE VALUE ArbitraryUnits of Biological Activity (units/100 pg) Chicken triple (M62I, I40F,I44L) ~9.9 M117any* ~1 M36T ~0.75 M36A ~0.75 M36I ~2.5 Avonex ~5.3 WtIFN beta ~1.2 HEK 293 transfection supernatants were retested, andrepeat transfections were tested. IFN beta activity of supernatant orAvonex was measured as inhibition of Daudi cell proliferation. Units/100pg are expressed as relative to wt IFN beta (1 unit/100 pg). *anynaturally occurring amino acid.

TABLE 5 BIOASSAY OF Met SUBSTITUTIONS AND monoMET IFN BETA APPROXIMATEVALUE Concentration (pg/ml) 10 ~45 ~65 ~90 ~200 ~500 ~800 ~2000 ~6000~8000 10000 ~40000 Anonex 96 115 105 104 103 91 69 61 61 50 55 35 newstd Avonex 92 92 100 111 98 81 61 52 51 51 54 41 new std repeat wtIFN-beta 83 92 115 101 105 90 70 59 52 48 55 41 KG1-50.1 wt IFN-beta 6592 100 101 99 89 65 52 47 45 48 31 KG1-52.1 Triple 60 79 71 52 51 58 5150 49 53 53 39 Wt 66 97 99 62 71 50 50 42 43 43 48 30 triple-M117S 51 7161 50 36 41 42 43 40 43 46 25 triple-M117T 48 56 39 33 36 38 41 43 43 4245 26 M36A-triple 58 69 51 56 35 33 41 31 30 39 47 26 M36T-triple 68 7271 59 51 48 48 43 43 49 50 34 Triple 59 80 70 56 55 51 59 55 50 54 54 39M36T-triple- 62 90 68 57 54 43 42 53 49 47 48 30 M117T M36T-triple- 7899 101 74 64 56 58 55 50 41 40 32 M117S Wt 68 99 102 86 71 65 49 47 4645 47 29

Example 17 ADDITIONAL IFNβ MUTEINS

In addition or instead of the previously disclosed peptide mutations,human interferon-β was modified by substituting glutamate for the serineat amino acid position 2, and serine for the cysteine at amino acidposition 17 of the naturally occurring peptide. The substitution atamino acid position 2 provided, among other advantages, increasedretention of the amino terminal amino acid substitution (methionineanalog) which, in some cases was azidohomoalanine. The substitution atamino acid position 17 provided, among other advantages, improvedpurification of protein produced in host cells, in particular E. coli.

Example 18 SUBSTITITON OF N-TERMINAL METHIONINE IN IFNβ

The sole remaining methionine in the mutant interferon-β molecules ofthe previous Example is the methionine at amino acid position 1. Thismethionine residue was replaced with a non-natural amino acid residue(azidohomoalanine or homoproparglyglycine). One method of replacing orsubstituting the methionine is by a fermentation process wherein thenon-natural amino acid is supplied in place of or at much higherconcentrations than the corresponding natural amino acid residue (inthis case, methionine) and using endogenous tRNA machinery. Anothermethod of substituting the methionine is by using an external mutantamino acid tRNA synthetase, and/or an external mutant tRNA molecule.Other methods may be used. The methionine substitution may be conductedin a host cell, such as E. coli, Pseudomonas, or mammalian cells. Themutant interferon-β molecule was expressed in an E. coli host cell.

In other instances, a host cell with a mutant amino terminal methionineaminopeptidase may be used to process or retain a non-natural aminoacid. In this fashion, a host cell harborning a mutant methionineaminopeptidase whose specificity has been altered with respect to thepenultimate amino acid residue is used for expression of the protein.Use of a secretion system in the host cell (such as E. coli) may usesignal peptidases and/or proteases that are present in the periplasm tocontrol the expression and processing of the amino terminal amino acid.

Example 19 PEGYLATION OF MODIFIED IFNβ

Upon substitution of a non-natural amino acid residue with themethionine at position 1, a chemical moiety (polyethylene glycol) wasconjugated to the residue. Since the amino acid position 1 is at theterminus, the attached chemical moiety had minimal interference with theprotein folding, overall structure and/or function. The chemical moietywas attached by way of a copper-catalyzed cycloaddition between an azideand an alkyne, but may be attached by other methods known in the artand/or described in other Examples (such as Example 6 or Example 7), orother areas of the present application.

Interferon-β constructs with specific methionine substitutions werescreened using transient transfections in mammalian cells (HEK 293 Tcells) and the supernatants analyzed as measured by, for example,anti-viral activity, anti-proliferative activity, and/or ELISA.

Example 20 STABILIZATION OF MODIFIED IFNβ

In addition to or instead of other amino acid substitutions disclosedherein, the serine amino acid at position 2 of the naturally occurringhuman interferon-β was modified to glutamate, and the cysteine at aminoacid position 17 was modified to serine. These substitutionssurprisingly provide increased stabilization and/or production of themodified proteins in the host cell.

Retention of the non-natural amino acid residue (such asazidohomoalanine or homoproparglycine) at the amino terminus duringprotein processing is necessary for addition of the chemical moiety(such as pegylation), and depends on the identity of the amino acidresidue at the penultimate residue position.

In other instances, it may be desirable for the non-natural amino acidresidue to be removed during protein processing, such as for allowingregulation of the location of amino acid substitutions. For example,efficient removal of the substitution of the amino terminal methionineof human interferon β with a non-natural amino acid residue (such asazidohomoalanine or homoproparglycine) allows for the introduction of amethionine analog in positions other than the amino terminus of themolecule, while retaining at least one non-natural amino acid residue inthe molecule.

In this regard, we found the highest retention of the non-natural aminoacid residue (such as azidohomoalanine or homoproparglycine) at theamino terminus of human interferon 13 when the penultimate amino acidresidue is selected from the following: glutamine, glutamic acid, orhistidine. We would also expect high retention when the penultimateamino acid residue of any protein is phenylalanine, methionine, lysine,tyrosine, tryptophan, or arginine. We found some retention of thenon-natural amino acid residue (such as azidohomoalanine orhomoproparglycine) when the penultimate amino acid residue is glycine orserine, and a low level of retention (high level of removal) of thenon-natural amino acid residue when the penultimate amino acid isalanine.

Example 21 MONOMET IFNβ

In one particular mutant of human interferon-β, MonoMet (which includeda single methionine replaced at the amino terminus during fermentationwith AHA, and with all other methionines replaced genetically), themutant protein was expressed in E. coli with either serine, alanine,glycine, glutamine, histidine or glutamic acid at amino acid positionnumber 2. When the amino acid at position 2 was serine, and the aminoterminal methionine was substituted with a non-natural amino acid(azidohomoalanine or homoproparglycine), the non-natural amino acid isnot efficiently retained and is partially processed, resulting inheterogenous protein products. Such products included proteins withuncleaved non-natural amino acids at the amino terminus, proteins withcleaved non-natural amino acids at the amino terminus, and proteins withformylated non-natural amino acids at the amino terminus. When the aminoacid at position 2 is histidine, glutamine or glutamic acid, the aminoterminal non-natural amino acid is highly retained.

When azidohomoalanine is used as the non-natural amino acid at the aminoterminus and the amino acid at position 2 of the human interferon β ishistidine, glutamine or glutamic acid, the azide moiety of theazidohomoalanine is retained and the N formyl group is removed.

When the amino acid at position 2 of the human interferon β is alanine,and the amino terminus methionine is substituted with a non-naturalamino acid (azidohomoalanine or homoproparglycine), the non-naturalamino acid is removed.

In addition to the non-natural amino acids used, other non-natural aminoacids may be incorporated instead, such as azidonorleucine.

A mutant interferon-β product was thus generated with AHA incorporatedat the amino terminus, and the other mutations are S2E, C17S, M36I,140F, I44L, M62I, M117T. The mutant interferon-β containing these aminoacid substitutions retained the amino terminal AHA, was easily purifiedand refolded properly (including disulfide bond formation).Additionally, the interferon-β mutant was efficiently PEGylated and thefinal formulation was stable and retained full biological activity bothin vitro and in vivo.

Example 22 PURIFICATION AND PEGYLATION OF PROTEINS AND INTERFERON-β BYCOPPER-CATALYZED AZIDE-ALKYNE CYCLOADDITION

We demonstrate a modified copper-catalyzed cycloaddition method forpegylation of a target molecule, such as a protein or peptide thatcontains a non-natural amino acid residue. The modified method allowsfor efficient purification, folding and oxidation of the targetmolecule. Typically, other methods of copper-catalyzed cycloadditionrequire the presence of Cu(I) by using ultrapure CuBr or CuSO₄ and areducing agent, such as TCEP or Cu(O). Our modified method is conductedin the presence of DTT. Without wishing to be bound to any particulartheory, the DTT may act either as a reducing agent for a biomoleculeand/or for the copper species, and may act as a ligand for copper in themodified cycloaddition reaction.

Oxygen may be required for the modified cycloaddition reaction,especially in the presence of reducing agents, and can be providedeither by introducing air into the reaction vessel or by allowing thereaction vessel to remain open to the ambient air, or by otherwiseadding oxidants and/or reductants to control the overall redox state ofthe reaction mixture. The modified cycloaddition reaction may beperformed by using non-natural amino acid-containing biomolecules,including reactions with or without a triazole linkage, and variousconcentrations of several copper species, SDS (which is desirable incertain embodiments), DTT, TCEP, and PEG-alkyne.

The reaction may occur in mixed micelle “microreactors” containing thetarget molecule and other reactants. The reaction may be sonicated,which may improve mass transport between different mixed micelles forimproved mixing, and/or affect the introduction of oxygen to thereaction mixture, as well as the mixture of copper oxidation states. Insome instances, subjecting the solubilized target molecule to afreeze/thaw cycle prior to beginning the reaction improves the CuBrcatalyzed reaction. The freeze/thaw cycle may affect mixed micelles ofthe target molecule, or otherwise affect solubility of the molecule. Inour modified method, the cycloaddition reaction is performedpreferentially using CuSO₄, rather than CuBr. Alkyne-PEGs may bemanufactured from PEG-NHS esters, either in organic or aqueous solution.

Modified interferon-β and PEG-interferon-β were purified by firstrefolding the interferon-β by dilution into a buffer containingzwittergent with no additional SDS, which allows for subsequent ionexchange chromatography analysis of the solution. Anion exchangechromatography and size exclusion chromatography may be used forpurifying pegylated and unpegylated interferon-β. The zwittergent may beremoved from the pegylated interferon-β while also removing unpegylatedinterferon-β. This allows for production of a pure pegylatedinterferon-β suitable for in vitro or in vivo assays or for clinicaladministration.

Example 23 PEG-INTERFERON-BETA INHIBITS TUMOR PROGRESSION IN MURINEXENOGRAFT MODEL

We tested the efficacy of PEG-(20K) interferon beta and its ability toinhibit the growth of a tumor grown subcutaneously in immunocompromised(SCID) mice compared to BETASERON®. PEG-(20K) interferon beta inhibitstumor progression in vivo more efficiently than BETASERON®.

Animal Studies

The mice used in these studies were female C.B-17 SCID mice 6-8 weeks.(Charles River Laboratories, Wilmington, Mass.). Food and water wereprovided ad libitum. Test animals were housed in a specificpathogen-free environment and allowed to acclimate in a temperature andhumidity controlled environment prior to the commencement ofexperimental procedures.

Daudi cells, a human B lymphoblastoid cell ine (ATCC, Manassas, Va.),were injected subcutaneously in the abdominal midline. Mice were treatedeither with PEG-(20K)-interferon (IFN) (3 U), BETASERON® (humaninterferon-β-1b) (10 U) or vehicle either once per week or three timesper week, following tumor implantation. After tumors became palpable(about 3 weeks) tumor measurements were made in two dimensions threetimes a week using digital calipers. Tumor volume was determined usingthe formula for a prolate spheroid. Tumor progression was measured for65 days.

Activity Studies

IFN beta was PEGylated and purified. The PEG IFN beta was compared tocommercial BETASERON® (Bayer Corp.) for antiviral activity using EC₅₀ asa measure of drug potency. The results are shown in FIGS. 8A and 8B. Thedata were analyzed using one way repeated measures ANOVA with aTukey-Kramer multiple comparison post test.

Example 24 Prophetic EXOGENOUS TRNA EXPRESSION PRODUCES DIFFERENTIALREGULATION OF GENES DUE TO CODON BIAS

It has previously been shown in eukaryotic cells that levels oftranslation of specific target genes can be altered by providing asingle tRNA expression construct. The authors suggested that the levelsor amount of tRNAs in cells is related to the levels of gene expressionat translation levels, and suggested that low levels of specific tRNAslead to low levels of translation potentially due to problems indecoding the mRNA in host cells containing large numbers of thespecified codons. See Gu, et al. Nuc. Acids Res. 32:4448 (2004), herebyincorporated by reference in its entirety. For example, if a particularhost cell contained high levels of a specific tRNA species, this highlevel of tRNA may result in codon bias of mRNA molecules for the majorprotein products of the cell. Thus, how a codon is used is approximatelyequal to the ability of the tRNA to regulate expression of the targetgenes in both differentiated and non-differentiated epithelium.

Considering this, using methods described herein, inter alia, it may bedesirable to use the cell's tendency for codon bias (i.e.“bias codon”)to specify an incorporation of a non-natural amino acid by introducingan exogenous or external mutant tRNA that decodes the bias codon and isaminoacylated by an exogenous or external mutant M-RS.

Example 25 SITE-SPECIFIC PEGYLATION OF INTERFERONβ BY CU(I)-CATALYZEDCYCLOADDITION

The present example demonstrates improved methods for site-specificPEGylation of proteins. In this study, nonnatural amino acids(s) wereused with a recombinant protein to produce site-specific conjugation.Site-specific PEGylation enhanced pharmacokinetic and pharmacodynamicproperties of the therapeutic protein including an increase in serumhalf-life and a decrease in bioactivity. In contrast, methods thatrandomly PEGylate proteins may yield a product protein that does notcontain the PEG in a specific location or site. Randomly PEGylatedprotein may have reduced bioactivity and/or produce a nonhomogeneousproduct and/or produce a product that varies from batch to batch.

mPEG-NHS 10 kDa linear, 20 kDa linear, and 40 kDa branched werepurchased from Nektar, San Francisco, Calif. and NOF, Tokyo Japan.PEG-alkyne were obtained from NOF, Tokyo Japan. Both AHA and triazoleligand were custom synthesized by MedChem Source, Federal Way, Wash.Copper sulfate and copper bromide (ultrapure) were obtained from SigmaAldrich, USA.

Methods to Produce IFNβ-AHA

Human IFNβ was mutated to remove 3 of 4 natural Met residues (M36I,M62I, M117T) (see: Ser. No. 11/743,608), to maintain bioactivity bymodifying two other residues that interact with the internal Met (I40F,I44L), and to eliminate methionine aminopeptidase cleavage of theN-terminal AHA by modifying the penultimate residue (S2E). TheN-terminal codon for Met in the mature protein was left intact.

Methionine auxotrophic host E. coli cells were used for proteinexpression. An auxotrophic host cell refers to a cell that is unable tosynthesize one or more natural amino acids. The cells were expanded inmedia containing all 20 natural amino acids, then washed and resuspendedin media containing 19 natural amino acids, AHA, and no Met. Followinginduction, the resulting recombinant protein contained AHA at theN-terminus instead of Met.

PEGylation was conducted according to the scheme diagrammed below.PEG-alkyne was reacted with IFNβ-AHA while the protein was denatured andreduced.

Three different mPEGs (either 10 kDa, 20 kDa or 40 kDa) werefunctionalized with an alkyne group by nucleophilic addition ofpropargylamine. NHS esters of PEG 10 kDa linear or 20 kDa linear PEG, or40 kDa branched were reacted with propargylamine in dioxane, as shownbelow.

mPEG-C2-NHS (10 kDa linear)+propargylamine→PEG-alkyne 10 k linear

mPEG-C5-NHS (20 kDa linear)+propargylamine→PEG-alkyne 20 k linear

mPEG₂-C3-NHS (40 kDa branched)+propargylamine→PEG-alkyne 40 k branched

Each PEG-NHS ester was dissolved in dioxane at 2.5 wt %, under argon.The reaction flasks were briefly placed in a warm water bath to dissolvethe PEG, with stirring. 50 molar excess (10K or 20K) or 100 molar excess(40 kDa PEG,) of propargylamine was added to each reaction. The mixtureswere allowed to react for at least 1.5 h at room temperature underargon. The functionalized PEG was isolated by dripping into 5-10× coldhexane, with stirring for 15 min, and then filtering through a 0.2 umnylon membrane. The crude precipitate was then dissolved in water with0.5-1 mL 1M NaOH to hydrolyze any remaining NHS ester (10 min) and thenneutralized with an equal volume of 1M HCl. The product was thendialyzed against 0.1M NaCl, 0.05M NaCl, and then water three times,followed by filtration and lyophilization. The product structure wasconfirmed by 1H NMR.

A schematic diagram of the procedure used for 10 kDa PEGylation of IFNβis provided below.

IFNAHA was reacted with mPEG-alkyne 10 kDa linear with varyingconcentrations of PEG, copper, and triazole ligand, and for varioustimes. The reaction occurred in 100 mM phosphate, 10 mM dithiothreitol,2% SDS, pH 7.55 buffer in an air atmosphere. The triazole ligand wasprepared as a stock in DMSO and the CuBr was prepared as a stock in H2O.The default reaction conditions were 0.5 mg/mL IFNAHA, 2 wt % PEG, 2 mMtriazole ligand, 1 mM CuBr, reacting for 24 h at room temperature.Increasing the PEG concentration to 2 mM and using a triazole ligand tocopper ratio of 1 or 2 produced the most PEG-IFN. Increasingconcentrations of copper (holding the triazole to copper ratio at 2)caused loss of protein from the reaction mixtures, especially at 2 mM orhigher.

Schematic diagrams of the procedures used for PEGylation of IFNI3 with20 kDa and 40 kDa PEG are shown below.

20 kDa Example

40 kDa Example

IFNAHA was reacted with mPEG-alkyne 20 kDa linear and 40 kDa branchedusing similar methods as those for the 10 kDa reaction. However, the 20kDa reaction occurred in 0.5% SDS, with 2 mg/mL IFNAHA, and 1 mM CuSO4(rather than CuBr), for 25 hours. The 40 kDa reaction occurred with 2 mMDTT and 8 mM oxidized DTT, with 1 mg/mL IFNAHA, and 1 mM CuSO4 (ratherthan CuBr), for 22 hours.

The reaction was optimized by weight-percent PEG-alkyne, concentrationof copper catalyst, and the molar ratio of triazole ligand to coppercatalyst. Soluble fractions of the reaction mixtures were separated bySDS-PAGE and the resulting PEG-IFNβ bands were quantified bydensitometry.. PEGylation was most efficient around 2-3 wt % PEG-alkyne.We noted that higher concentrations were less efficient and hypothesizethat efficiency of pegylation may be limited by viscosity. We noted thata 1:1 or 2:1 molar ratio of triazole ligand to copper catalyst was mostefficient.

Higher concentrations of CuBr caused the protein (primarily theunPEGylated protein) to precipitate, perhaps by copper-induced oxidationreactions. Optimal conditions resulted in >90% of IFNβ-AHA beingPEGylated.

The PEG20 k-IFNβ ran around 60 k Mr and the PEG40 k-IFNβ ran around 140k Mr on SDS-PAGE. 50% of the IFNAHA reacted with PEG20 kDa and 57% ofthe IFNAHA reacted with PEG40 kDa.

Utilizing the procedures described above, site-specific PEGylation ofIFNAHA was achieved. This PEGylation was specific to the AHA residue atposition 1, with no side reaction to other residues.

Bioactivities of the various PEGylated IFNAHAs and Betaseron® (acommercially available IFNβ-1β; Bayer HealthCare Pharmaceuticals) wereassessed in vitro as the ability to prevent lysis of A549 cells exposedto EMC virus (lower EC50=more potent in vitro). The results are shownbelow.

Betaseron®: EC₅₀=17 pg/mL

PEG10-IFNβ: EC₅₀=11 pg/mL

PEG20-IFNβ: EC₅₀=48 pg/mL

PEG40-IFNβ: EC₅₀=1249 pg/mL

In vitro bioactivity of the PEG-IFNps was a function of the MW of theconjugated PEG.

This study demonstrates that site-specific PEGylated IFNβ produced bythe methods described herein retains biological activity. These methodsallow PEGylation of recombinant proteins at a single pre-determinedsite, without side reactions to other residues within the protein. Theresulting PEGylated proteins retain biological activity and are expectedto have enhanced pharmacokinetic properties, including increased serumhalf-life.

Example 26 REFOLDING OF EXPRESSED PROTEINS

This example provides methods for obtaining refolded, soluble forms ofproteins, and in this particular example, to refold a PEGylated protein.We describe a method for increasing the yield of a PEGylated protein byusing the methods described herein to refold PEG-beta interferon.

The methods are generally accomplished by preparing a refolded, solubleform of an insoluble or aggregated PEG interferon beta protein whichcontains one or more free cysteine residues. The methods include thesteps of: a) causing a host cell to express a interferon beta protein inan insoluble or aggregated form; b) lysing the cells by chemical,enzymatic or physical means; c) solubilizing the insoluble or aggregatedprotein by exposing the insoluble or aggregated protein to a denaturingagent, a reducing agent and a cysteine blocking agent; and d) PEGylatingthe reduced, denatured protein, and e) refolding the PEGylated proteinby reducing the concentrations of the denaturing agent and reducingagents to levels sufficient to allow the PEG interferon beta protein torefold into a soluble, biologically active form.

Examplary procedures and reagents are described below.

Protein Solution

0.1-0.5 mg/ml IFNβ-PEG

20 mM NaPhosphate pH7.5

150 mM NaCl

10 mM DTT

1 mM EDTA

0.02% SDS

Refolding Buffer

10 mM Tris pH10

10 mM NaCl

1 mM EDTA

6 μM CuSO4

0.3% Zwittergent 3-14

The Protein Solution and Refolding Buffer are used in a range ofconcentrations in relation to one another. For example, the proteinconcentration in the Protein Solution is effective in a wide range ofconcentrations, e.g., between 0.001-20 mg/ml or, preferably, 0.1-0.5mg/ml in a preferred range. In addition, any buffer, e.g., Tris, Hepes,Acetate, citrate, etc.may be used at a pH range 2-12. In this example,the preferred pH is 7.5 or in a range of pH 6.0-9.0. The NaClconcentration in the protein solution may range between 0 to 2 M orpreferably 150 mM. DTT is added in a concentration range between 0 to200 mM between 100 μM and 20 mM (preferably 10 mM). The concentration ofDTT must be adjusted depending on the concentration of CuSO₄ used duringrefolding using a stochiometric ratio between 100-250 fold excess DTT toCuSO₄. The EDTA concentration can range between 0 and 50 mM or 100 μMand 1 mM. As with the concentration of DTT, the concentration of EDTA isadjusted depending on the concentration of CuSO₄ during refolding usinga stochiometric ratio between 100-250 fold excess EDTA to CuSO₄aspreviously described. In addition, the concentration of SDS may rangebetween 0 and 1% or in the range of 0.01 and 0.1%.

With respect to the Refolding Buffer, Tris buffer provided the mostefficient protein refolding, with a pH of at least 10 is required forcertain proteins. NaCl concentration can range between 0 and 2M with apreferred concentration of 1-50 mM. The EDTA concentration can rangebetween 0 and 50 mM or between 100 μM and 2 mM; with a preferredconcentration of EDTA of 1 mM. As previously described, thestoichometric ratio of 100-250 fold excess EDTA to CuSO₄ are requiredduring refolding.

Other salts of Cu can be used and other metals beside Cu can be used,i.e., Pd, Pt, Au, Ag, etc.

CuSO₄ (6 μM) or in the range of 4-10 μM was found to be the optimalconcentration when used with 1 mM EDTA. However, as previously describedthe appropriate concentration is dependent on the DTT and EDTAconcentrations, and will generally be in the range of 0.1-100 μM or 4-10μM in a preferred range.

Zwittergent 3-14 surfactant can range between 0.1 and 1.0% and can besubstituted by other non-denaturing surfactants such as Tween-80,octylglucoside, etc. or by denaturing surfactants at concentrations lowenough to allow refolding such as SDS.

Protocol:

Protein refolding is performed at room temperature by following thefollowing steps:

1) Drip Protein Solution into rapidly mixing (>200 rpm) 10-fold (or inthe range of 5-20 fold) volume of Refolding Buffer. The Refolding Buffervolume can range between 0.5-500-fold or in the range of 1-20-fold orpreferably 5-10 fold dilution of the Protein Solution volume. ProteinSolution may be provided to the refolding buffer in pulses withrefolding time between pulses.

2) Adjust pH to 10.0 with 5M NaOH (several drops are required). Otherbuffers or bases may be used for pH adjustment.

3) Mix for 2.5 hours, 22° C., 150 rpm. In some instances, mixing at pH10 may range between 10 minutes to 48 hours with a preferred time of 2-3hours.

4) Adjust pH to 5.0 by adding 1M Acetic Acid to a final concentration of20 mM. Other buffers or acids besides acetic acid can be used to reducethe pH to 5.0. pH can range between 2 to 12,

These methods may be utilized to achieve proper folding of recombinantproduced proteins, including the site-specific mutants described herein.Accordingly, these methods provide bioactive proteins.

Example 27 SITE-SPEClFIC PEGYLATION OF INTERFERONβ BY CU(I)-CATALYZEDCYCLOADDITION

The present example demonstrates additional compounds and methods forsite-specific PEGylation of proteins. A branched PEG-alkyne (40 kDa) wasobtained from NOF, Tokyo Japan (NOF Product No. GL2-400PP2). Each branchof the PEG-portion of the PEG-alkyne was 20 kDa. Both AHA and triazoleligand were custom synthesized by MedChem Source (Federal Way, Wash.).Copper sulfate and copper bromide (ultrapure) were obtained from SigmaAldrich, USA. A modified human interferon-β polypeptide (IFNβ-AHA) wasprepared according the procedures disclosed above comprising thefollowing amino acid alterations: methionine at position 1 toazidohomoalanine, serine at position 2 to glutamic acid, cysteine atposition 17 to serine, methionine at position 36 to isoleucine,isoleucine at position 40 to phenylalanine, isoleucine at position 44 toleucine, methionine at position 62 to isoleucine, and methionine atposition 117 to threonine.

PEGylation at position 1 the modified human interferon-β polypeptide(IFNβ-AHA) was conducted according to the scheme diagrammed below.PEG-alkyne was reacted with IFNβ-AHA while the protein was denatured andreduced.

In the above reaction, PEG-alkyne was reacted with IFNβ-AHA byCu(I)-catalyzed azide and alkyne cycloaddition. The reactants werecombined at the final concentrations shown below, with the triazoleadded second to last and the copper added last. The triazole ligand wasadded as a solid to the reaction.

IFNb-AHA 1 mg/mL PEG-Alkyne 0.6 wt % PEG diol (30 kDa) 1.4 wt % SDS 2%DTT 2 mM Phosphate (pH 7.55) 80 mM Triazole ligand 3 mM CuSO₄ 1.5 mM

The PEG diol and triazole ligand had the following structures. Thereaction was prepared at 1 L scale in a 2 L beaker. The reactionproceeded overnight with mixing at room temperature. The PEGylationefficiency was assessed by SDS-PAGE. The reaction proceeded to 71%PEG-IFNβ.

After the above PEGylation reaction PEG-IFNβ was captured on ceramichydroxyapatite (CHT), eluted and folded, captured on cation ion exchange(IEX) and the eluant concentrated for polish step on size exclusionchromatography (SEC). The CHT column separated PEGylated IFNb from thepegylation reaction mix components and from the residual unpegylatedIFNβ. Protein from the CHT column was rapidly diluted into a 4C buffercontaining 1% Tween-80 and trace amounts of CuSO₄ balanced with DTT andEDTA. The pH was adjusted to 10.2 and the protein allowed to foldovernight at 4C. After folding the pH was reduced to 4.0 and the samplewas applied to an IEX column. The IEX column served to exchange thesurfactant from SDS to Tween-80 and to concentrate protein forsubsequent SEC polish step. Eluant from the IEX column was concentratedby ultrafiltration prior to application to the SEC column. The SECcolumn separated aggregate from monomer and exchanged buffer into finalformulation buffer. The eluted target protein was concentrated, sterilefiltered and packaged to provide PEG-IFNβ.

In vivo potency of the above PEG-IFNβ was demonstrated by its ability toinhibit growth of Daudi cell tumors in scid mice, generally according toprocedures described in Example 23. 10⁷ Daudi cells were implantedsubcutaneously in scid mice. Treatments were begun on the day ofimplantation and terminated after 4 weeks of treatment. Animals weresacrificed when tumors reached 1000 mm³. The results of this assay arepresented in FIG. 9 at different levels of administration, along withcomparative data against buffer [what was buffer?] and Betaseron.

The in vitro efficacy of the above PEG-IFNβ as compared to Betaseron wasdemonstrated by assaying their ability to inhibit the cytopathic effectof EMC virus on A549 cells, according to procedures described in Example23. The results of this assay are presented in FIG. 10, which shows thattwo different preparations of PEG-IFNb were capable fof inhibiting EMCvirus.

Example 28 IN VIVO PHARMACOKINETIC PROFILE OF PEG-IFNβ

The pharmacokinetic characteristics of the PEG-IFNβ of Example 27 wereexamined in cynomolgus monkeys in comparison to Avonex®, a recombinantInterferon beta-1a.

PEG-IFNβ was administered to male cynomolgus monkeys via a singleintravenous injection (0.06 mg/kg) in a saphenous vein or a subcutaneousinjection (0.3 mg/kg) in the mid-scapular region. Avonex® wasadministered via intravenous injection or intramuscular injection. Threeanimals were used for each treatment group. Plasma was collected fromeach animal via a femoral vein at the following time points: pre-doseand at 0.17 (intravenous groups only), 0.5, 2, 4, 6, 8, 10, 12, 18, 24,48, 72, 96, 120, and 144 hours post-dose. PEG-IFNβ and Avonex® levelswere measured using an A549 antiviral assay, as shown in FIG. 11 for asingle subcutaneous administration of PEG-IFNβ. During the study,animals were observed twice daily for mortality and signs of pain anddistress. In addition, cage side observations for general health andappearance were done once daily.

The pharmacokinetic parameters of PEG-IFNβ and Avonex® followingsubcutaneous, intramuscular or intravenous dosing including AUC_(all),AUClNF, t_(1/2), Cl, V_(c), Vss, Tmax, and Cmax were obtained from thenon-compartmental analysis of the plasma data using WinNonlin and isprovided in Tables 6 and 7. The calculated bioavailability wasdetermined by calculating the ratio of the area under the curve from thetime of dosing extrapolated to infinity (AUC(_(0-∞))) to compareparameters between intravenous and subcutaneous injection. Afterintravenous injection, the mean serum PEG-IFNβ concentration declinedover time in a biphasic manner with a terminal elimination half-life(t½) of 29.77 hours. The volume of distribution (V_(z)) was 73.81 ml/kg,indicating that the drug was in the more accessible compartment.

TABLE 6 Pharmacokinetic Profiles of PEG-IFNβ in Cynomolgus Monkeys Calc.Route of Animal Cmax Tmax t_(1/2) AUC_((all)) AUC_((0-∞)) V_(z) CIBioavail- Delivery # (ng/mL) (h) (h) (h*ng/mL) (h*ng/mL) (mL/kg)(mL/kg/h) ability (%) IV C20091 NA NA 28.86 35,725 37,861 65.99 1.58A01567 NA NA 25.60 32,064 34,033 65.11 1.76 C21561 NA NA 34.84 30,56233,392 90.32 1.80 Average NA NA 29.77 32,783 35,095 73.81 1.71 SC C200891,241.07 10.00 34.52 63,240 68,276 NA NA C22405 954.86 8.00 56.40 49,65459,111 NA NA C22354 537.18 8.00 79.47 37,591 52,877 NA NA Average 911.048.67 56.80 50,162 60,088 NA NA 34.2

TABLE 7 Pharmacokinetic Profiles of Avonex ® in Cynomolgus Monkeys Calc.Route of Animal Cmax Tmax t_(1/2) AUC_((all)) AUC_((0-∞)) V_(z) CIBioavail- Delivery # (ng/mL) (h) (h) (h*ng/mL) (h*ng/mL) (mL/kg)(mL/kg/h) ability (%) IV C20637 NA NA 4.0 91.28 91.7 473.04 81.8 C20076NA NA 4.9 119.1 120.7 440.24 62.1 C21597 NA NA 4.0 218.9 220.4 199.4934.0 Average NA NA 4.3 143.1 144.3 370.9 59.3 IM C20637 34.2 2 3.8 190.0194.3 NA NA C20076 47.3 2 4.9 307.5 307.8 NA NA C21597 15.9 2 5.8 346.6348.2 NA NA Average 34.5 2 4.8 281.4 283.4 NA NA 98.2

Following subcutaneous injection of PEG-IFNβ, a longer absorption phaseand a lower level of peak concentration was achieved, as compared tointravenous injection. The average half-life (t½) of 57 hours wasgreater in comparison to intravenous injection, indicating that the rateof clearance was substantially decreased. The average bioavailability ofPEG-IFNβ was approximately 34%. No adverse clinical observations werenoted during the study with either 0.06 mg/kg intravenous dosing or the0.3 mg/kg subcutaneous dosing. These results demonstrate that PEG-IFNβhas a much greater half-life in comparison to Avonex®, indicating thatthe PEGylated interferon-β is cleared at a much slower rate.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific method and reagents described herein, including alternatives,variants, additions, deletions, modifications and substitutions. Suchequivalents are considered to be within the scope of this invention andare covered by the following claims.

1. A method for producing a modified target polypeptide, comprising: (a)providing a host cell, the host cell comprising a vector having apolynucleotide encoding the target polypeptide, (b) site-specificallyincorporating one or more non-natural amino acid codons into thepolynucleotide, wherein at least one non-natural amino acid codoncorresponds to the first position of the amino terminus of the targetpolypeptide, (c) growing the host cell under conditions such that thehost cell expresses the modified target polypeptide, wherein themodified target polypeptide comprises a non-natural amino acid residueat the first position of the amino terminus, and wherein the non-naturalamino acid residue comprises an azide, alkyne, vinyl, or aryl halidegroup, thereby producing a modified target polypeptide.
 2. The method ofclaim 1 wherein at least one non-natural amino acid codon corresponds tothe penultimate position of the amino terminus of the modified targetpolypeptide.
 3. The method of claim 1 wherein the one or morenon-natural amino acids is selected from the group consisting of:azidonorleucine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine,p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine,m-ethynyl-phenylalanine, 6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid,p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine,p-acetylphenylalanine, 3-(6-chloroindolyl)alanine,3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine,homopropargylglycine, p-chlorophenylalanine, α-aminocaprylic acid,O-methyl-L-tyrosine, N-acetylgalactosamine-α-threonine, andN-acetylgalactosamine-α-serine.
 4. The method of claim 1 furthercomprising attaching a chemical moiety to one or more of the non-naturalamino acid residues in the modified target polypeptide.
 5. The method ofclaim 4 wherein the chemical moiety is attached to the non-natural aminoacid residue at the first position of the amino terminus of the modifiedtarget polypeptide.
 6. The method of claim 4 wherein the chemical moietyis attached to the non-natural amino acid residue by a covalentinteraction, a single carbon-carbon linkage, a double carbon-carbonlinkage, a triple carbon-carbon linkage, or a triazole linkage betweenthe non-natural amino acid and the chemical moiety.
 7. The method ofclaim 4 wherein the chemical moiety is attached to the non-natural aminoacid residue by way of a chemical reaction selected from the groupconsisting of: copper catalyzed [3+2] cycloaddition, Suzuki coupling,Hiyama coupling, Kumada coupling, Heck reaction, Cadiot-Chodkiewiczcoupling, and Sonogashira coupling.
 8. The method of claim 4 wherein thechemical moiety is selected from the group consisting of: cytotoxins,pharmaceutical drugs, dyes, fluorescent labels, a nucleophilic group, anelectrophilic group, a ketone, an aldehyde, an azide, an alkyne,photocaged groups, tags, a peptide, a polypeptide, a protein, anoligosaccharide, a polyethylene glycol of any molecular weight and inany geometry, a polyvinyl alcohol, a polyaklylene oxide, metals, metalcomplexes, polyamines, imidazoles, carbohydrates, lipids, biopolymers,particles, solid supports, a polymer, a water-soluble polymer, atargeting agent, an affinity group, any agent to which a complementaryreactive chemical group can be attached, biophysical probes, biochemicalprobes, isotypically-labeled probes, spin-label amino acids,fluorophores, aryl iodides and bromides.
 9. The method of claim 1wherein the target polypeptide is selected from the group consisting of:α-1 antitrypsin, Angiostatin, Antihemolytic factor, an antibody, anantibody fragment, an antibody derivative, a Fab, aFab═, aF(ab)2, a Fd,a Fv, a ScFv, a diabody, a tribody, a tetrabody, a dimer, a trimer, aminibody, an angiogenic molecule, an angiostatic molecule,Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, an Atrial peptide, a C—X—C chemokine,Calcitonin, a CC chemokine, CD40 ligand, C-kit ligand, Chloramphenicalacetyltransferase, a Collagen, a Colony stimulating factor, Complementfactor 5a, Complement inhibitor, Complement receptor 1, a cytokine, anEpidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B,Factor IX, Factor VII, Factor VIII, Factor X, a Fibroblast GrowthFactor, Fibrinogen, Fibronectin, Follitropin, G-CSF, GM-CSF,Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein,Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hirudin, Human serum albumin, Human GrowthHormone, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF,INF-α, IFN-β, IFN-γ, IFN-Ω, IFN-τ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemiainhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor,oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF,peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin,Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin,SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukinreceptors, Soluble TNF receptor, soluble adhesion molecules,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen,Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors,Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator,Tumor necrosis factor β, Tumor necrosis factor receptor, Tumor necrosisfactor-α, Vascular Endothelial Growth Factor, a virus-like particle,Urokinase; a transcriptional modulator that modulates cell growth,differentiation, or regulation, an expression activator, an inflammatorymolecule, a growth factor, a growth factor receptor, an oncogeneproduct, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD4OL/CD40, VLA-4NCAM-1, ICAM-1/LFA-1, hyalurin/CD44, asignal transduction molecule, a transcriptional activator, atranscriptional suppressor, estrogen, progesterone, testosterone,aldosterone, LDL, corticosterone amidase, amino acid racemase, anacylase, a dehalogenase, a dioxygenase, a diarylpropane peroxidase, anepimerase, epoxide hydrolase, an esterase, an isomerase, a kinase, aglucose isomerase, a glycosidase, a glycosyl transferase, ahaloperoxidase, a monooxygenase, a lipase, a lignin peroxidase, nitrilehydratase, nitrilase, protease, a phosphatase, subtilisin, atransaminase, and a nuclease.
 10. The method of claim 1 wherein thesite-specifically incorporating one or more non-natural amino acidcodons is conducted by a technique selected from the group consistingof: site-directed mutagenesis, error-prone PCR, gene shuffling,homologous recombination, incorporation of an amber stop codon,incorporation of a wobble codon, use of an external mutantaminoacyl-tRNA synthetase, and incorporation of a bias codon.
 11. Themethod of claim 9 wherein the target polypeptide comprises interferon-β.12. The method of claim 11 wherein at least one of the non-natural aminoacid codons corresponds to positions selected from the group consistingof: 2, 17, 36, 40, 44, 62, and 117 of the modified target polypeptide.13. A composition comprising a modified target polynucleotide encoding amodified target polypeptide, the modified target polynucleotidecomprising one or more non-natural amino acid codons that encode atleast one non-natural amino acid comprising an azide, alkyne, vinyl, oraryl halide group and corresponding to the first position of the aminoterminus of the target polypeptide.
 14. The composition of claim 13further comprising a host cell.
 15. The composition of claim 13 whereinat least one non-natural amino acid codon corresponds to the penultimateposition of the amino terminus of the target polypeptide.
 16. Thecomposition of claim 13 wherein the modified target polypeptide isselected from the group consisting of: α-1 antitrypsin, Angiostatin,Antihemolytic factor, an antibody, an antibody fragment, an antibodyderivative, a Fab, aFab′, aF(ab)2, a Fd, a Fv, a ScFv, a diabody, atribody, a tetrabody, a dimer, a trimer, a minibody, an angiogenicmolecule, an angiostatic molecule, Apolipoprotein, Apoprotein, Atrialnatriuretic factor, Atrial natriuretic polypeptide, an Atrial peptide, aC—X—C chemokine, Calcitonin, a CC chemokine, CD40 ligand, C-kit ligand,Chloramphenical acetyltransferase, a Collagen, a Colony stimulatingfactor, Complement factor 5a, Complement inhibitor, Complement receptor1, a cytokine, an Epidermal Growth Factor, Erythropoietin, Exfoliatingtoxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, aFibroblast Growth Factor, Fibrinogen, Fibronectin, Follitropin, G-CSF,GM-CSF, Glucocerebrosidase, Gonadotropin, growth factor, Hedgehogprotein, Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus,Hepatitis B virus, Hepatitis C virus, Hirudin, Human serum albumin,Human Growth Hormone, Insulin, Insulin-like Growth Factor, GM-CSF,G-CSF, M-CSF, INF-α, IFN-β, IFN-γ, IFN-Ω, IFN-τ, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin,leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitoryfactor, oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF,PDGF, peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin,Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin,SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukinreceptors, Soluble TNF receptor, soluble adhesion molecules,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen,Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors,Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator,Tumor necrosis factor β, Tumor necrosis factor receptor, Tumor necrosisfactor-α, Vascular Endothelial Growth Factor, a virus-like particle,Urokinase; a transcriptional modulator that modulates cell growth,differentiation, or regulation, an expression activator, an inflammatorymolecule, a growth factor, a growth factor receptor, an oncogeneproduct, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD4OL/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, asignal transduction molecule, a transcriptional activator, atranscriptional suppressor, estrogen, progesterone, testosterone,aldosterone, LDL, corticosterone amidase, amino acid racemase, anacylase, a dehalogenase, a dioxygenase, a diarylpropane peroxidase, anepimerase, epoxide hydrolase, an esterase, an isomerase, a kinase, aglucose isomerase, a glycosidase, a glycosyl transferase, ahaloperoxidase, a monooxygenase,a lipase, a lignin peroxidase, nitrilehydratase, nitrilase, protease, a phosphatase, subtilisin, atransaminase, and a nuclease.
 17. A modified target polypeptidecomprising one or more non-natural amino acid residues, wherein at leastone of the non-natural amino acid residues is the first position of theamino terminus, wherein the non-natural amino acid residue comprises anazide, alkyne, vinyl, or aryl halide group.
 18. The modified targetpolypeptide of claim 17 further comprising a chemical moiety attached toone or more non-natural amino acid residues in the target polypeptide.19. The modified target polypeptide of claim 18 wherein the chemicalmoiety is attached at least to the non-natural amino acid residue in thefirst position of the amino terminus of the target polypeptide.
 20. Themodified target polypeptide of claim 18 wherein the chemical moiety isattached to the non-natural amino acid corresponding to the firstposition of the amino terminus of the target polypeptide by a covalentattachment, a single carbon-carbon linkage, a double carbon-carbonlinkage, a triple carbon-carbon linkage, or a triazole linkage betweenthe chemical moiety and the non-natural amino acid.
 21. The modifiedtarget polypeptide of claim 18 wherein the chemical moiety is selectedfrom the group consisting of: cytotoxins, pharmaceutical drugs, dyes,fluorescent labels, a nucleophilic group, an electrophilic group, aketone, an aldehyde, an azide, an alkyne, photocaged groups, tags, apeptide, a polypeptide, a protein, an oligosaccharide, a polyethyleneglycol of any molecular weight and in any geometry, a polyvinyl alcohol,a polyaklylene oxide, metals, metal complexes, polyamines, imidazoles,carbohydrates, lipids, biopolymers, particles, solid supports, apolymer, a water-soluble polymer, a targeting agent, an affinity group,any agent to which a complementary reactive chemical group can beattached, biophysical probes, biochemical probes, isotypically-labeledprobes, spin-label amino acids, fluorophores, aryl iodides and bromides.22. The modified target polypeptide of claim 17 wherein the targetpolypeptide is selected from the group consisting of: α-1 antitrypsin,Angiostatin, Antihemolytic factor, an antibody, an antibody fragment, anantibody derivative, a Fab, aFab′, aF(ab)2, a Fd, a Fv, a ScFv, adiabody, a tribody, a tetrabody, a dimer, a trimer, a minibody, anangiogenic molecule, an angiostatic molecule, Apolipoprotein,Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide,an Atrial peptide, a C—X—C chemokine, Calcitonin, a CC chemokine, CD40ligand, C-kit ligand, Chloramphenical acetyltransferase, a Collagen, aColony stimulating factor, Complement factor 5a, Complement inhibitor,Complement receptor 1, a cytokine, an Epidermal Growth Factor,Erythropoietin, Exfoliating toxins A and B, Factor IX, Factor VII,Factor VIII, Factor X, a Fibroblast Growth Factor, Fibrinogen,Fibronectin, Follitropin, G-CSF, GM-CSF, Glucocerebrosidase,Gonadotropin, growth factor, Hedgehog protein, Hemoglobin, HepatocyteGrowth Factor, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hirudin, Human serum albumin, Human Growth Hormone, Insulin,Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF, INF-α, IFN-β, IFN-γ,IFN-Ω, IFN-τ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,Keratinocyte Growth Factor, Lactoferrin, leukemia inhibitory factor,Luciferase, Neurturin, Neutrophil inhibitory factor, oncostatin M,Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF, peptidehormones, Phenylalanine hydroxylase, Urikase, Pleiotropin, Protein A,Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, SCF, Solublecomplement receptor I, Soluble I-CAM 1, Soluble interleukin receptors,Soluble TNF receptor, soluble adhesion molecules, Somatomedin,Somatostatin, Somatotropin, Streptokinase, Superantigen, Staphylococcalenterotoxin, Superoxide dismutase, Toll-like receptors, Toxic shocksyndrome toxin, Thymosin α1, Tissue plasminogen activator, Tumornecrosis factor β, Tumor necrosis factor receptor, Tumor necrosisfactor-α, Vascular Endothelial Growth Factor, a virus-like particle,Urokinase; a transcriptional modulator that modulates cell growth,differentiation, or regulation, an expression activator, an inflammatorymolecule, a growth factor, a growth factor receptor, an oncogeneproduct, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, asignal transduction molecule, a transcriptional activator, atranscriptional suppressor, estrogen, progesterone, testosterone,aldosterone, LDL, corticosterone amidase, amino acid racemase, anacylase, a dehalogenase, a dioxygenase, a diarylpropane peroxidase, anepimerase, epoxide hydrolase, an esterase, an isomerase, a kinase, aglucose isomerase, a glycosidase, a glycosyl transferase, ahaloperoxidase, a monooxygenase,a lipase, a lignin peroxidase, nitrilehydratase, nitrilase, protease, a phosphatase, subtilisin, atransaminase, and a nuclease.
 23. A pharmaceutical compositioncomprising a modified target polypeptide of claim
 17. 24. A method forpreparing a refolded, soluble form of an insoluble or aggregatedPEGylated interferon beta protein comprising one or more free cysteineresidues, said method comprising the steps of: (a) causing a host cellto express a interferon beta protein in an insoluble or aggregated form;(b) lysing the cells by chemical, enzymatic or physical means; (c)solubilizing the insoluble or aggregated protein by exposing theinsoluble or aggregated protein to a denaturing agent, a reducing agentand a cysteine blocking agent; (d) PEGylating the reduced, denaturedprotein; and (e) refolding the PEGylated protein by reducing theconcentrations of the denaturing agent and reducing agents to levelssufficient to allow the PEG interferon beta protein to refold into asoluble, biologically active form.
 25. A method for producing a modifiedtarget polypeptide, comprising: (a) providing a host cell comprising avector, wherein said vector comprises a polynucleotide encoding a targetpolypeptide; (b) site-specifically incorporating one or more non-naturalamino acid residue(s) into the target polypeptide to produce a modifiedtarget polypeptide, wherein: (i) one or more codons encoding naturallyoccurring amino acids are incorporated into said polynucleotide; (ii)the one or more non-natural amino acid(s) are incorporated into thetarget polypeptide at one or more natural amino acid position(s) encodedby the one or more codon(s) of (i) or one or more unchanged codon(s)encoding a natural amino acid; and (c) growing the host cell underconditions such that the host cell expresses the modified targetpolypeptide.
 26. The method of claim 25, wherein the modified targetpolypeptide comprises one or more non-natural amino acid(s)site-specifically incorporated into the target polypeptide at one ormore natural amino acid position(s) corresponding to (i).
 27. The methodof claim 25, wherein said site-specific incorporation of (b) occurs byprotein translation in the host cell, and wherein said proteintranslation occurs either by mutant transcription machinery or mutanttranslation machinery, or both.
 28. The method of claim 27, wherein saidmutant transcription machinery comprises one or more mutant tRNA(s)and/or mutant amino-acyl tRNA synthetase(s).
 29. The method of claim 25,wherein the one or more non-natural amino acid residues(s) of (ii)replace(s) one or more methionine, tryptophan, isoleucine or leucineresidue.
 30. The method of claim 25, wherein the non-natural amino acidresidue(s) comprise(s) a side chain having one or more functionalgroup(s) selected from the group consisting of: bromo-, iodo-, ethynyl-,cyano-, azido-, acetyl, aryl ketone, ketone, aldeyhyde, alkenyl-, anazide group, an alkyne group, a vinyl group, an aryl halide group, aphotolabile group, a fluorescent group, and a heavy metal.
 31. Themethod of claim 25, wherein the one or more non-natural amino acidresidue(s) is selected from the group consisting of: azidonorleucine,3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine,p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine,6-ethynyl-tryptophan, 5-ethynyl-tryptophan,(R)-2-amino-3-(4-ethynyl-1H-pyrol-3-yl)propanic acid,p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine,p-acetylphenylalanine, 3-(6-chloroindolyl)alanine,3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine,homopropargylglycine, p-chlorophenylalanine, α-aminocaprylic acid,O-methyl-L-tyrosine, N-acetylgalactosamine-α-threonine, andN-acetylgalactosamine-α-serine.
 32. The method of claim 25, furthercomprising attaching one or more chemical moieties to one or more ofsaid one or more non-natural amino acid residue(s).
 33. The method ofclaim 32, wherein the one or more chemical moieties is selected from thegroup consisting of: cytotoxins, pharmaceutical drugs, dyes, fluorescentlabels, a nucleophilic group, an electrophilic group, a ketone, analdehyde, an azide, an alkyne, photocaged groups, tags, a peptide, apolypeptide, a protein, an oligosaccharide, a polyethylene glycol of anymolecular weight and in any geometry, a polyvinyl alcohol, apolyaklylene oxide, metals, metal complexes, polyamines, imidazoles,carbohydrates, lipids, biopolymers, particles, solid supports, apolymer, a water-soluble polymer, a targeting agent, an affinity group,any agent to which a complementary reactive chemical group can beattached, biophysical probes, biochemical probes, isotypically-labeledprobes, spin-label amino acids, fluorophores, aryl iodides and bromides.34. A method of modifying a polypeptide containing a non-natural aminoacid, comprising incorporating a chemical moiety into said polypeptideby a chemical reaction selected from the group consisting of:palladium-catalyzed Suzuki coupling, palladium-catalyzed Sonogashiracoupling, palladium-catalyzed Heck coupling, palladium-catalyzed Hiyamacoupling and Huisgen copper-catalyzed [3+2] cycloaddition, in thepresence of micelles or DTT, or both, wherein said modified polypeptideretains its native function.
 35. The method of claim 34, wherein saidchemical moiety comprises a functional group selected from the groupconsisting of: an alkyne, an azide, an alkene, a boronic acid, a silane,an aryl halide, a vinyl halide, and an alkenyl halide,
 36. The method ofclaim 34, wherein said chemical moiety comprises a moiety selected fromthe group consisting of: a PEG, a polymer, a water soluble polymer, anoligosaccharide, a toxin, a drug, and a polypeptide.
 37. The method ofclaim 34, wherein the target polypeptide is selected from the groupconsisting of: α-1 antitrypsin, Angiostatin, Antihemolytic factor, anantibody, an antibody fragment, an antibody derivative, a Fab, aFab′,aF(ab)2, a Fd, a Fv, a ScFv, a diabody, a tribody, a tetrabody, a dimer,a trimer, a minibody, an angiogenic molecule, an angiostatic molecule,Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, an Atrial peptide, a C—X—C chemokine,Calcitonin, a CC chemokine, CD40 ligand, C-kit ligand, Chloramphenicalacetyltransferase, a Collagen, a Colony stimulating factor, Complementfactor 5a, Complement inhibitor, Complement receptor 1, a cytokine, anEpidermal Growth Factor, Erythropoietin, Exfoliating toxins A and B,Factor IX, Factor VII, Factor VIII, Factor X, a Fibroblast GrowthFactor, Fibrinogen, Fibronectin, Follitropin, G-CSF, GM-CSF,Glucocerebrosidase, Gonadotropin, growth factor, Hedgehog protein,Hemoglobin, Hepatocyte Growth Factor, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hirudin, Human serum albumin, Human GrowthHormone, Insulin, Insulin-like Growth Factor, GM-CSF, G-CSF, M-CSF,INF-α, IFN-β, IFN-γ, IFN-Ω, IFN-τ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, IL-29, Keratinocyte Growth Factor, Lactoferrin, leukemiainhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor,oncostatin M, Osteogenic protein, Parathyroid hormone, PD-ECSF, PDGF,peptide hormones, Phenylalanine hydroxylase, Urikase, Pleiotropin,Protein A, Protein G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin,SCF, Soluble complement receptor I, Soluble I-CAM 1, Soluble interleukinreceptors, Soluble TNF receptor, soluble adhesion molecules,Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigen,Staphylococcal enterotoxin, Superoxide dismutase, Toll-like receptors,Toxic shock syndrome toxin, Thymosin a 1, Tissue plasminogen activator,Tumor necrosis factor β, Tumor necrosis factor receptor, Tumor necrosisfactor-α, Vascular Endothelial Growth Factor, a virus-like particle,Urokinase; a transcriptional modulator that modulates cell growth,differentiation, or regulation, an expression activator, an inflammatorymolecule, a growth factor, a growth factor receptor, an oncogeneproduct, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD4OL/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, asignal transduction molecule, a transcriptional activator, atranscriptional suppressor, estrogen, progesterone, testosterone,aldosterone, LDL, corticosterone amidase, amino acid racemase, anacylase, a dehalogenase, a dioxygenase, a diarylpropane peroxidase, anepimerase, epoxide hydrolase, an esterase, an isomerase, a kinase, aglucose isomerase, a glycosidase, a glycosyl transferase, ahaloperoxidase, a monooxygenase, a lipase, a lignin peroxidase, nitrilehydratase, nitrilase, protease, a phosphatase, subtilisin, atransaminase, and a nuclease.
 38. A modified human interferon-13polypeptide comprising the following amino acid alterations: methionineat position 1 to azidohomoalanine, serine at position 2 to glutamicacid, cysteine at position 17 to serine, methionine at position 36 toisoleucine, isoleucine at position 40 to phenylalanine, isoleucine atposition 44 to leucine, methionine at position 62 to isoleucine, andmethionine at position 117 to threonine.
 39. The polypeptide of claim38, wherein said azidohomoalanine is conjugated to a polyalkyleneglycol.
 40. The polypeptide of claim 39, wherein the polyalkylene glycolhas a molecular weight of from about 1,000 Daltons to about 100 kDa. 41.The polypeptide of claim 38, wherein the polyalkylene glycol has amolecular weight of from about 2 kDa to about 60 kDa.
 42. Thepolypeptide of claim 38, wherein the polyalkylene glycol has a molecularweight of from about 10 kDa to about 40 kDa.
 43. The polypeptide ofclaim 38, wherein the polyalkylene glycol is polyethylene glycol and hasa molecular weight of about 40 kDa.
 44. The polypeptide of claim 38,wherein the polyalkylene glycol is unbranched.
 45. The polypeptide ofclaim 38, wherein the polyalkylene glycol is branched.
 46. Thepolypeptide of claim 45, wherein the branched polyalkylene glycolcomprises two branches each with a molecular weight of about 18 kDa toabout 22 kDa.
 47. The polypeptide of claim 46, wherein the branchedpolyalkylene glycol is a branched polyethylene glycol.
 48. Thepolypeptide of claim 38, wherein the polyalkylene glycol is conjugatedto the polypeptide through a triazole linkage.
 49. A compositioncomprising the polypeptide of claim 38, and a pharmaceuticallyacceptable diluent or excipient.